Abstract: Due to their central role in cellular fat storage and lipid homeostasis, lipid droplets (LD) have attracted great interest in biomedical research. The integration of both biochemical and genetic tools and the use of model organisms have greatly contributed to the understanding of LD metabolism and its regulation. However, many important aspects such as LD biogenesis, intracellular dynamics, or their potential degradation by autophagy are still poorly understood. Microscopic techniques, in particular fluorescence microscopy using LD specific dyes or fluorescent protein tagging, represent excellent experimental tools to study the dynamic nature of both the protein and lipid content of LD. Single cell systems in culture are particularly suited to identify and characterize proteins required for LD formation and turnover, using genetic knock-down or gene deletion strategies. Here we describe experimental setups to investigate LD dynamics and turnover in yeast, using various labeling techniques suitable for three-dimensional imaging over time (4D imaging), quantitative microscopy and imaging-based screens of mutant libraries. Also, implementation of coherent anti-Stokes Raman scattering (CARS) microscopy as an emerging tool for label-free lipid imaging in living cells will be discussed.
Abstract: Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time-lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro LD (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of bovine serum albumin (BSA) in the culture medium that acts as a fatty acid (FA) scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor Triacsin C implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering (CARS) microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several hours and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular FA overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.
Abstract: Rationale: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support. Objective: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. Methods and Results: Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. Conclusions: The data indicate that vascular bioactivation of GTN is catalyzed by cytosolic ALDH2. Mitochondrial GTN metabolism may contribute to oxidative stress-related adverse effects of nitrate therapy and the development of nitrate tolerance.
Abstract: Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.
Abstract: Malfunctions of processes involved in cellular lipid storage and mobilization induce the pathogenesis of prevalent human diseases such as obesity, type 2 diabetes and atherosclerosis. Lipid droplets are the main lipid storage depots for neutral lipids in eukaryotic cells, and as such fulfil an essential function to balance cellular lipid metabolism and energy homeostasis. Despite significant progress in identifying key metabolic enzymes involved in lipid storage and their regulation in various model organisms, some fundamental questions as to the biogenesis, subcellular distribution and inheritance of lipid droplets are as yet unsolved. In this study, we applied a set of imaging techniques such as high-resolution four-dimensional (4D) live-cell imaging, quantitative microscopy, transmission electron microscopy and electron tomography to gain insight into the spatio-temporal organization of lipid droplets during cellular growth in the yeast Saccharomyces cerevisiae. This analysis revealed a high level of organization of the subcellular positioning of lipid droplets in individual cells, their directed migration towards the cellular periphery and a coordinated transfer of a subpopulation of lipid droplets into daughter cells during cell division. Lipid droplets appear to remain associated with ER membranes during cellular growth independently of their size and subcellular localization. Deletion of FLD1, the functional orthologue of the human BSCL2 gene encoding seipin, leads to impaired dynamics of yeast lipid droplets and defective lipolysis, which might be due to aberrant ER structures in these mutants. Our data suggest a role for yeast seipin as a scaffolding protein that is required for the dynamics of a specific subdomain of the ER, and provide a new aspect for the interpretation of abnormal lipid droplets phenotypes in yeast mutants lacking seipin.
Abstract: Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino(-) phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino(-) phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino(-) phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells.
Abstract: Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.
Abstract: Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae.
Abstract: Lipotoxicity is the pathological consequence of lipid overflow in non-adipose tissue, mediated through reactive lipid moieties which may even lead to lipid-induced cell death (lipoapoptosis). This derailment of cellular and organismal fat homeostasis is the consequence of obesity due to continued over-feeding, and contributes substantially to the pathogenesis of insulin resistance, type 2 diabetes mellitus and cardiovascular disease, which are all components of the metabolic syndrome. Now, does yeast, a single-celled eukaryote, ever suffer from the metabolic syndrome and what can we potentially learn from studies in this organism about the underlying molecular mechanism that lead to lipid-associated pathologies in human cells? In this review I will summarize the remarkably conserved metabolic and regulatory processes relevant to establishing cellular energy and lipid homeostasis, as well as recent findings that provide detailed insights into the molecular mechanisms underlying fat-induced cellular malfunction and cell death, with potential implications also for mammalian cells.
Abstract: The endemic increase in lipid-associated disorders such as obesity and type 2 diabetes mellitus has placed triacylglycerol metabolism and its associated organelle, lipid droplets, in the spotlight of biomedical research. Key enzymes of triacylglycerol metabolism are structurally and functionally conserved between yeast and mammalian cells, and studies in yeast have contributed significantly to the understanding of their biological function(s). Based on these similarities, studies performed in yeast may provide further significant mechanistic insight into the molecular basis of triacylglycerol homeostasis and its important physiological roles in healthy and diseased cells.
Abstract: BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161-Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.
Abstract: Obesity is characterised by lipid accumulation in non-adipose tissues, leading to organ degeneration and a wide range of diseases, including diabetes, heart attack and liver cirrhosis. Free fatty acids (FFA) are believed to be the principal toxic triggers mediating the adverse cellular effects of lipids. Here, we show that various cooking oils used in human nutrition cause cell death in yeast in the presence of a triacylglycerol lipase, mimicking the physiological microenvironment of the small intestine. Combining genetic and cell death assays, we demonstrate that elevated FFA concentrations lead to necrotic cell death, as evidenced by loss of membrane integrity and release of nuclear HMGB1. FFA-mediated necrosis depends on functional mitochondria and leads to the accumulation of reactive oxygen species. We conclude that lipotoxicity is executed via a mitochondrial necrotic pathway, challenging the dogma that the adverse effects of lipid stress are exclusively apoptotic.
Abstract: Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Delta mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.
Abstract: Biological membranes with cubic morphology are a hallmark of stressed or diseased cellular conditions; both protein-protein interactions and lipid alterations appear to contribute to their biogenesis, yet their specific cellular functions are unknown. The occurrence of cubic membranes strikingly correlates with viral infections; notably, virus entry, proliferation, and release are processes closely linked to cellular cholesterol metabolism, and dys-regulation of cholesterol synthesis at the level of HMG-CoA reductase also induces cubic membrane formation, in the absence of viral infection. We propose that virus-induced cubic membranes could result from viral interference of cellular cholesterol homeostasis, generating a protective membrane environment to facilitate virus assembly and proliferation. Preventing cubic membrane formation might thus disrupt the 'virus factory' and offer new avenues to combat viral infections.
Abstract: In mammals, excess energy is stored in the form of triacylglycerol primarily in lipid droplets of white adipose tissue. The first step of lipolysis (i.e. the mobilization of fat stores) is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58), and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a three-dimensional homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (amino acids 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein. (1)H NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Although wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that the ability of CGI-58 to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.
Abstract: We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.
Abstract: Biological membranes are among the most fascinating assemblies of biomolecules: a bilayer less than 10 nm thick, composed of rather small lipid molecules that are held together simply by noncovalent forces, defines the cell and discriminates between "inside" and "outside", survival, and death. Intracellular compartmentalization-governed by biomembranes as well-is a characteristic feature of eukaryotic cells, which allows them to fulfill multiple and highly specialized anabolic and catabolic functions in strictly controlled environments. Although cellular membranes are generally visualized as flat sheets or closely folded isolated objects, multiple observations also demonstrate that membranes may fold into "unusual", highly organized structures with 2D or 3D periodicity. The obvious correlation of highly convoluted membrane organizations with pathological cellular states, for example, as a consequence of viral infection, deserves close consideration. However, knowledge about formation and function of these highly organized 3D periodic membrane structures is scarce, primarily due to the lack of appropriate techniques for their analysis in vivo. Currently, the only direct way to characterize cellular membrane architecture is by transmission electron microscopy (TEM). However, deciphering the spatial architecture solely based on two-dimensionally projected TEM images is a challenging task and prone to artifacts. In this review, we will provide an update on the current progress in identifying and analyzing 3D membrane architectures in biological systems, with a special focus on membranes with cubic symmetry, and their potential role in physiological and pathophysiological conditions. Proteomics and lipidomics approaches in defined experimental cell systems may prove instrumental to understand formation and function of 3D membrane morphologies.
Abstract: The mitochondrial dimeric phospholipid cardiolipin is characterized by a high degree of unsaturation of its acyl chains, which is important for its functional interaction with mitochondrial enzymes. The unusual fatty acid composition of cardiolipin molecular species emerges from a de novo synthesized "premature" species by extensive acyl chain remodeling that involves as yet only partially identified acyltransferases and phospholipases. Recently, the yeast protein Taz1p was shown to function as a transacylase, which catalyzes the reacylation of monolysocardiolipin to mature cardiolipin. A defect in the orthologous human TAZ gene is associated with Barth syndrome, a severe genetic disorder, which may lead to cardiac failure and death in childhood. We now identified the protein encoded by reading frame YGR110W as a mitochondrial phospholipase, which deacylates de novo synthesized cardiolipin. Ygr110wp has a strong substrate preference for palmitic acid residues and functions upstream of Taz1p, to generate monolysocardiolipin for Taz1p-dependent reacylation with unsaturated fatty acids. We therefore rename the Ygr110wp as Cld1p (cardiolipin-specific deacylase 1).
Abstract: Very long-chain polyunsaturated fatty acids (VLC-PUFAs), such as docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), have recently made it to the realm of "magical molecules" based on their multiple presumably beneficial effects in biological systems, making these PUFAs particularly interesting in biomedicine. Their specific biological functions, however, remain enigmatic. Here we provide evidence derived from studies in the amoeba Chaos that indicates a structural role for omega-6 DPA in cell membrane organization, which may help to explain the multiple diverse effects of VLC-PUFA in healthy and diseased states. Amoeba Chaos mitochondria undergo a remarkable and reversible morphological transition into cubic morphology on starvation. This morphological transition is reflected in major changes in fatty acid and lipid composition, as determined by gas liquid chromatography and mass spectrometry, in particular by a drastic increase in C22:5 modified phosphatidylcholine plasmalogen, phosphatidylethanolamine plasmalogen, and phosphatidylinositol species. Liposomes produced in vitro from lipids of starved amoeba cells show a high propensity to form hexagonal tubular and cubic morphologies. Addition of omega-6 DPA, but not of omega-3 DPA, to the cell culture also induced mitochondrial membrane transformation into cubic morphology in fed cells, demonstrating for the first time an important structural role of omega-6 DPA-containing lipids in cell membrane organization.
Abstract: Despite its small size of 5-8 mum - only one order of magnitude above the wavelength of visible light - yeast has developed into an attractive system for light microscopic analysis. First, the ease of genetic manipulation and integrative transformation have opened numerous experimental strategies for genome-wide tagging approaches, e.g., with fluorescent proteins (as discussed in several chapters of this issue). Second, the large number of cells that can be simultaneously visualized provides an excellent basis for statistical image analysis, resulting in reliable morphological or localization information. Third, the flexibility of yeast cultivation in terms of biochemical manipulation, rapid cellular growth, mutant isolation or drug susceptibility offers an unprecedented spectrum of possibilities for in vivo functional studies, and analysis of cellular dynamics and organelle inheritance. Although yeast in itself is an interesting cellular system, its "prototype character" in understanding cellular metabolism, physiology, and signaling in eukaryotes accounts for its popular use in technology development and biomedical research.Here we discuss experimental strategies for live yeast cell imaging, geared towards imaging-based large-scale screens. Major emphasis is on the methods for immobilizing cells under "physiological" conditions, with minimum impact on yeast. We also point out potential pitfalls resulting from live cell imaging that once again stresses the necessity for extremely careful experimental design and interpretation of data resulting from imaging experiments. It goes without saying that these problems are not restricted to yeast and are also highly relevant to "large" cells. If an image tells more than a thousand (perhaps misleading?) words, the ease of obtaining "images" thus rather suggests analyzing many thousands of images, to come up with one relevant and biologically significant conclusion.
Abstract: Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with beta-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl beta-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.
Abstract: Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.
Abstract: Triacylglycerols (TGs) serve essential cellular functions as reservoirs for energy substrates (fatty acids) and membrane lipid precursors (diacylglycerols and fatty acids). Here we show that the major yeast TG lipase Tgl4, the functional ortholog of murine adipose TG lipase ATGL, is phosphorylated and activated by cyclin-dependent kinase 1 (Cdk1/Cdc28). Phospho-Tgl4-catalyzed lipolysis contributes to early bud formation in late G1 phase of the cell cycle. Conversely, lack of lipolysis delays bud formation and cell-cycle progression. In the absence of beta-oxidation, lipolysis-derived metabolites are thus required to support cellular growth. TG homeostasis is the only metabolic process identified as yet that is directly regulated by Cdk1/Cdc28-dependent phosphorylation of key anabolic and catabolic enzymes, highlighting the importance of FA storage and mobilization during the cell cycle. Our data provide evidence for a direct link between cell-cycle-regulatory kinases and TG degradation and suggest a general mechanism for coordinating membrane synthesis with cell-cycle progression.
Abstract: Neuropathy target esterase (NTE) is a member of the family of patatin domain-containing proteins and exhibits phospholipase activity in brain and cultured cells. NTE was originally identified as target enzyme for organophosphorus compounds that cause a delayed paralyzing syndrome with degeneration of nerve axons. Here we show that the structurally related murine protein NTE-related esterase (NRE) is a potent lysophospholipase. The enzyme efficiently hydrolyzes sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid. No lipase activity was observed when triacylglycerols, cholesteryl esters, retinyl esters, phosphatidylcholine, or monoacylglycerol were used as substrates. Although NTE is predominantly expressed in the nervous system, we found the highest NRE mRNA levels in testes, skeletal muscle, cardiac muscle, and adipose tissue. Induction of NRE mRNA concentrations in these tissues during fasting suggested a nutritional regulation of enzyme expression and, in accordance with this observation, insulin reduced NRE mRNA levels in a dose-dependent manner in 3T3-L1 adipocytes. A green fluorescent protein-NRE fusion protein colocalized to the endoplasmic reticulum and lipid droplets. Thus, NRE is a previously unrecognized ER- and lipid droplet-associated lysophospholipase. Regulation of enzyme expression by the nutritional status and insulin suggests a role of NRE in the catabolism of lipid precursors and/or mediators that affect energy metabolism in mammals.
Abstract: Seeking to better understand how membrane trafficking is coordinated with phospholipid synthesis in yeast, we investigated lipid synthesis in several Sec(-) temperature-sensitive mutants, including sec13-1. Upon shift of sec13-1 cells to the restrictive temperature of 37 degrees C, phospholipid synthesis decreased dramatically relative to the wild type control, whereas synthesis of neutral lipids, especially triacylglycerol (TAG), increased. When examined by fluorescence microscopy, the number of lipid droplets appeared to increase and formed aggregates in sec13-1 cells shifted to 37 degrees C. Electron microscopy confirmed the increase in lipid droplet number and revealed that many were associated with the vacuole. Analysis of lipid metabolism in strains lacking TAG synthase genes demonstrated that the activities of the products of these genes contribute to accumulation of TAG in sec13-1 cells after the shift to 37 degrees C. Furthermore, the permissive temperature for growth of the sec13-1 strain lacking TAG synthase genes was 3 degrees C lower than sec13-1 on several different growth media, indicating that the synthesis of TAG has physiological significance under conditions of secretory stress. Together these results suggest that following a block in membrane trafficking, yeast cells channel lipid metabolism from phospholipid synthesis into synthesis of TAG and other neutral lipids to form lipid droplets. We conclude that this metabolic switch provides a degree of protection to cells during secretory stress.
Abstract: We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death.
Abstract: Triacylglycerol metabolism in Saccharomyces cerevisiae was analyzed quantitatively using a systems biological approach. Cellular growth, glucose uptake and ethanol secretion were measured as a function of time and used as input for a dynamic flux-balance model. By combining dynamic mass balances for key metabolites with a detailed steady-state analysis, we trained a model network and simulated the time-dependent degradation of cellular triacylglycerol and its interaction with fatty acid and membrane lipid synthesis. This approach described precisely, both qualitatively and quantitatively, the time evolution of various key metabolites in a consistent and self-contained manner, and the predictions were found to be in excellent agreement with experimental data. We showed that, during pre-logarithmic growth, lipolysis of triacylglycerol allows for the rapid synthesis of membrane lipids, whereas de novo fatty acid synthesis plays only a minor role during this growth phase. Progress in triacylglycerol hydrolysis directly correlates with an increase in cell size, demonstrating the importance of lipolysis for supporting efficient growth initiation.
Abstract: Lipid-associated disorders are a worldwide health concern and major efforts are directed toward understanding--at the molecular levels--mechanisms of lipid storage and degradation, in healthy and diseased states. Yeast is a widely used model organisms to study such processes at the cellular level because of significant functional and structural conservation of the factors involved in lipid metabolism. The focus of this study is on the microscopic investigation of the turnover of lipid droplets that are the intracellular storage compartments for fat, using a combination of green fluorescent protein-tagging and vital dye-labeling techniques. The applications and pitfalls of such techniques in understanding lipid storage and degradation are discussed.
Abstract: In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.
Abstract: To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.
Abstract: The mitochondrial phosphate carrier SLC25A3 transports inorganic phosphate into the mitochondrial matrix, which is essential for the aerobic synthesis of adenosine triphosphate (ATP). We identified a homozygous mutation--c.215G-->A (p.Gly72Glu)--in the alternatively spliced exon 3A of this enzyme in two siblings with lactic acidosis, hypertrophic cardiomyopathy, and muscular hypotonia who died within the 1st year of life. Functional investigation of intact mitochondria showed a deficiency of ATP synthesis in muscle but not in fibroblasts, which correlated with the tissue-specific expression of exon 3A in muscle versus exon 3B in fibroblasts. The enzyme defect was confirmed by complementation analysis in yeast. This is the first report of patients with mitochondrial phosphate-carrier deficiency.
Abstract: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes.
Abstract: Fatty acids are essential compounds in the cell. Since the yeast Saccharomyces cerevisiae does not feed typically on fatty acids, cellular function and growth relies on endogenous synthesis. Since all cellular organelles are involved in--or dependent on--fatty acid synthesis, multiple levels of control may exist to ensure proper fatty acid composition and homeostasis. In this review, we summarize what is currently known about enzymes involved in cellular fatty acid synthesis and elongation, and discuss potential links between fatty acid metabolism, physiology and cellular regulation.
Abstract: The 2001 Nobel Prize in Medicine, awarded to two yeast researchers for contributions to understanding the eukaryotic cell cycle, spotlighted yeast as an experimental model system in biomedical research. Major discoveries of molecular processes underlying lipid and biomembrane biogenesis were first made in yeast: secretory pathways, vesicle and membrane fusion, and the unfolded protein response. The discovery of programmed cell death that is conserved at multiple levels (quite intriguing for a unicellular organism), and energy metabolism controlled by adenosine monophosphate-activated protein kinases, mitogen-activated protein kinase signaling pathways, and the target of rapamycin (TOR) pathway (originally discovered in yeast)-all refer to functional and structural similarities with mammalian cells beyond the mere metabolic level. This article reviews recently uncovered aspects of fatty acid-associated malfunctions and lipotoxicity in yeast that may aid in understanding the molecular basis of lipid-associated disorders in mammals.
Abstract: Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.
Abstract: TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.
Abstract: We describe the design of a novel in vitro assay to study the interaction of soluble proteins with small hydrophobic sterol ligands. The sterol molecules are incorporated in an artificial membrane system in order to mimic their arrangement found in a biomembrane. The artificial membrane setup is monitored in real time by surface plasmon spectroscopy. Binding of fluorescently labeled soluble protein is observed by optical detection with surface plasmon enhanced fluorescence spectroscopy. By application of the novel assay, we demonstrate that four different oxidized sterol molecules are specifically recognized by the yeast protein Osh5p, a presumed oxysterol binding protein. Osh5p from yeast is the first oxysterol binding protein homologue for which oxysterol binding is shown with this new technique. With the design of our novel in vitro oxysterol binding assay, we have solved the technically challenging difficulty of presenting hydrophobic ligands to hydrophilic proteins in aqueous media.
Abstract: Storage and degradation of triglycerides are essential processes to ensure energy homeostasis and availability of precursors for membrane lipid synthesis. Recent evidence suggests that an emerging class of enzymes containing a conserved patatin domain are centrally important players in lipid degradation. Here we describe the identification and characterization of a major triglyceride lipase of the adipose triglyceride lipase/Brummer family, Tgl4, in the yeast Saccharomyces cerevisiae. Elimination of Tgl4 in a tgl3 background led to fat yeast, rendering growing cells unable to degrade triglycerides. Tgl4 and Tgl3 lipases localized to lipid droplets, independent of each other. Serine 315 in the GXSXG lipase active site consensus sequence of the patatin domain of Tgl4 is essential for catalytic activity. Mouse adipose triglyceride lipase (which also contains a patatin domain but is otherwise highly divergent in primary structure from any yeast protein) localized to lipid droplets when expressed in yeast, and significantly restored triglyceride breakdown in tgl4 mutants in vivo. Our data identify yeast Tgl4 as a functional ortholog of mammalian adipose triglyceride lipase.
Abstract: Cubic membranes represent highly curved, three-dimensional nanoperiodic structures that correspond to mathematically well defined triply periodic minimal surfaces. Although they have been observed in numerous cell types and under different conditions, particularly in stressed, diseased, or virally infected cells, knowledge about the formation and function of nonlamellar, cubic structures in biological systems is scarce, and research so far is restricted to the descriptive level. We show that the "organized smooth endoplasmic reticulum" (OSER; Snapp, E.L., R.S. Hegde, M. Francolini, F. Lombardo, S. Colombo, E. Pedrazzini, N. Borgese, and J. Lippincott-Schwartz. 2003. J. Cell Biol. 163:257-269), which is formed in response to elevated levels of specific membrane-resident proteins, is actually the two-dimensional representation of two subtypes of cubic membrane morphology. Controlled OSER induction may thus provide, for the first time, a valuable tool to study cubic membrane formation and function at the molecular level.
Abstract: The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.
Abstract: Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.
Abstract: The Yeast Protein Localization database (YPL.db(2)) is an archive of microscopic image data of protein localization patterns in the yeast Saccharomyces cerevisiae. The current version of YPL.db(2) harbours 500 sets of image data derived from high-resolution microscopic analyses of proteins tagged with the green fluorescent protein (GFP). Major functional improvements in YPL.db(2) over a previous release are a web-based experiment and image submission interface, facilitating standardized data entry by remote users through the Internet. The image display page provides image gallery and image scrolling features. In addition, fluorescence and transmission images can be superimposed, allowing image fading for precise correlation of the protein's localization in the cellular context. The reference structure database displaying 'prototypic' localization patterns was extended, and a feature to display and manipulate 3D-image datasets, using a freely available VRML plug-in, was included. Access to the Yeast Protein Localization database version 2.0 (YPL.db(2)) is available through http://YPL.uni-graz.at.
Abstract: Phosphatidylinositol (PtdIns) phosphates are involved in signal transduction, cytoskeletal organization, and membrane traffic. PtdIns 4-phosphate [PtdIns(4)P], produced in yeast by PtdIns 4-kinase (Pik1p), appears to regulate Golgi secretory function. PtdIns(4)P is also produced by dephosphorylation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], catalyzed by one of the three yeast Sjl proteins, homologs of the mammalian synaptic vesicle-associated PtdIns(4,5)P2 5-phosphatase, synaptojanin. To determine whether Pik1p and Sjl proteins operate in the same pathway or regulate the same process, we used a genetic approach. Mutation in the PIK1 gene displays synthetic genetic interactions with deletions of individual SJL genes. Deletion of SJL3 gene is synthetically lethal with pik1ts, and deletions of SJL1 or SJL2 genes in pik1ts cells exacerbate the temperature sensitivity, neomycin sensitivity, and defect in invertase secretion. A diminished level of PtdIns(4)P and increased level of PtdIns(4,5)P2 in pik1(ts)sjl1delta and pik1(ts)sjl2delta cells, compared with pik1ts cells, indicate that PtdIns(4)P is specifically required for secretion. Collectively, our results suggest that Pik1p and the Sjl proteins coordinately function to regulate the dynamic phosphorylation-dephosphorylation of the polar heads of phosphoinositides, and this process appears to be important for membrane trafficking pathways.
Abstract: S-adenosyl-L-homocysteine hydrolase (Sah1p, EC 3.3.1.1.) is a key enzyme of methylation metabolism. It catabolizes S-adenosyl-L-homocysteine, which is formed after donation of the activated methyl group of S-adenosyl-L-methionine (AdoMet) to an acceptor, and which acts as strong competitive inhibitor of all AdoMet-dependent methyltransferases. Sah1p is an essential enzyme in yeast and one of the most highly conserved proteins with up to 80% sequence homology throughout all kingdoms of life. SAH1 expression in yeast is subject to the general transcriptional control of phospholipid synthesis. Profound changes in cellular lipid composition upon depletion of Sah1p support the notion of a tight interaction between lipid metabolism and Sah1p function.
Abstract: We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.
Abstract: Adaptation of cells to acetic acid requires a hitherto unknown number of proteins. Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid. Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family). Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y. lipolytica. Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid. Expression of GPR1 is induced by acetic acid and moderately repressed by glucose. It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions. Fluorescence microscopy confirmed a localization to the plasma membrane. A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.
Abstract: Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p.
Abstract: Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.
Abstract: Large free-living amoeba (Chaos carolinensis) can survive in spring water without food intake for several weeks. Starvation is associated with a dramatic change in mitochondrial cristae from random tubular to ordered (paracrystalline) cubic morphology. Whole-cell polarography was used to monitor changes in respiratory activity during fasting. Basal respiration per cell decreased progressively during starvation, while the cyanide-resistant fraction increased. Spectrofluorometric assay of H2O2 and reactive oxygen species (ROS) in cell lysates (using the dye 2',7'-dichlorofluorescein diacetate) indicates greater H2O2 and ROS generation in starved than in fed cells. Fluorescence microscopy of intact cells incubated with the same dye demonstrates that H2O2 and ROS tend to accumulate in vacuoles. A remarkable generation of O2 observed with starved cells after addition of KCN may be explained by release of H2O2 from these compartments into the cytosol, where it can react with catalase. Together, these observations suggest that fasting increases oxidative stress in the amoeba and that this organism has several protective mechanisms to deal with it, including activation of a plantlike alternative oxidase. The hypothesis is forwarded that the cubic structural transition of the mitochondrial inner membrane represents another protective mechanism, reducing oxidative damage by enhancing the efflux of H2O2 and ROS and by reducing the susceptibility of membrane lipids to the oxidants.
Abstract: The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C. and Yaffe, M.P. (1991). J. Cell Biol. 115, 1249-1257). We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.
Abstract: The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitro elongation assays confirm that the ybr159Delta mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Delta mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein-tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesis is essential for viability, the ybr159Delta mutant cells are viable (albeit very slowly growing) and do synthesize some VLCFA. This suggested that a functional ortholog of Ybr159p exists that is responsible for the residual 3-ketoreductase activity. By disrupting the orthologs of Ybr159w in the ybr159Delta mutant we found that the ybr159Deltaayr1Delta double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.
Abstract: The Yeast Protein Localization database (YPL.db) contains information about the localization patterns of yeast proteins resulting from microscopic analyses. The data and parameters of the experiments to obtain the localization information, together with images from confocal or video microscopy, are stored in a relational database, building an archive of, and the documentation for, all experiments. The database can be queried based on gene name, protein localization, growth conditions and a number of additional parameters. All experiment parameters are selectable from predefined lists to ensure database integrity and conformity across different investigators. The database provides a structure reference resource to allow for better characterization of unknown or ambiguous localization patterns. Links to MIPS, YPD and SGD databases are provided to allow fast access to further information not contained in the localization database itself. YPL.db is available at http://ypl.tugraz.at.
Abstract: Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles. Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP. Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible. In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles. Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles. Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity.
Abstract: Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1Delta strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1Delta strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains. Together, these findings indicate that the inositol auxotrophy of snf1Delta strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.
Abstract: Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.
Abstract: The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Delta mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. The TSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons. These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3 gene, both of which have previously been shown to be required for VLCFA synthesis. Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in a tsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis. Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex. Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.
Abstract: The Saccharomyces cerevisiae ALR1 (YOL130w) gene product Alr1p is the first known candidate for a Mg(2+) transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg(2+) transporter family. Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg(2+). Using well characterized npi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis. Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole. Mutants lacking Alr1p (Deltaalr1) showed a 60% reduction of total intracellular Mg(2+) compared with the wild type and failed to grow in standard media. When starved of Mg(2+), mutant and wild-type cells had similar low levels of intracellular Mg(2+); but upon addition of Mg(2+), wild-type cells replenished the intracellular Mg(2+) pool within a few hours, whereas Deltaalr1 mutant cells did not. Expression of the bacterial Mg(2+) transporter CorA in the yeast Deltaalr1 mutant partially restored growth in standard media. The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg(2+).
Abstract: A molecular genetic characterization of the ORF YOR304W (ISW2), identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein, is presented in this paper. The protein encoded by the ORF YOR304W is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family and has been recently entered into SDG as ISW2. Although not essential for vegetative growth, we found that the ISW2 gene is required for early stages in sporulation. The isw2 homozygous deletant diploid strain was blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. ISW2 expression from a multicopy plasmid had the same effect as deletion, but ISW2 expression from a centromeric plasmid rescued the deletion phenotype. In vegetatively growing diploid cells, the Isw2 protein was preferentially found in the cytoplasm, co-localizing with microtubules. An accumulation of the Isw2 protein within the nucleus was observed in cells entering sporulation. Together with data published very recently by Tsukiyama et al. (1999), we propose a role for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes.
Abstract: In the yeast Saccharomyces cerevisiae, two acyl-CoA:sterol acyltransferases (ASATs) that catalyze the synthesis of steryl esters have been identified, namely Are2p (Sat1p) and Are1p (Sat2p). Deletion of either ARE1 or ARE2 has no effect on cell viability, and are1are2 double mutants grow in a similar manner to wild-type despite the complete lack of cellular ASAT activity and steryl ester formation [Yang, H., Bard, M., Bruner, D. A., Gleeson, A., Deckelbaum, R. J., Aljinovic, G., Pohl, T. M., Rothstein, R. & Sturley, S. L. (1996) Science 272, 1353-1356; Yu, C., Kennedy, J., Chang, C. C. Y. & Rothblatt, J. A. (1996) J. Biol. Chem. 271, 24157-24163]. Here we show that both Are2p and Are1p reside in the endoplasmic reticulum as demonstrated by measuring ASAT activity in subcellular fractions of are1 and are2 deletion strains. This localization was confirmed by fluorescence microscopy using hybrid proteins of Are2p and Are1p fused to green fluorescent protein (GFP). Lipid analysis of are1 and are2 deletion strains revealed that Are2p and Are1p utilize sterol substrates in vivo with different efficiency; Are2p has a significant preference for ergosterol as a substrate, whereas Are1p esterifies sterol precursors, mainly lanosterol, as well as ergosterol. The specificity towards fatty acids is similar for both isoenzymes. The lack of steryl esters in are1are2 mutant cells is largely compensated by an increased level of free sterols. Nevertheless, terbinafine, an inhibitor of ergosterol biosynthesis, inhibits growth of are1are2 cells more efficiently than growth of wild-type. In a growth competition experiment are1are2 cells grow more slowly than wild-type after several rounds of cultivation, suggesting that Are1p and Are2p or steryl esters, the product formed by these two enzymes, are more important in the natural environment than under laboratory conditions.
Abstract: A novel rapid polymerase chain reaction (PCR)-based technique for N-terminal attachment of green fluorescent protein (GFP) to a yeast protein is described. Genomic integration of a PCR-generated loxPkanMX4loxP-yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N-terminally to the protein of interest. In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site-specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon. The essential protein Ydl193wp of unknown function and the oleate-inducible fatty acid activation protein, encoded by FAA2, were N-terminally tagged using the novel technique. Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomally expressed GFP-Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid-borne GFP construct expressed under control of the MET25(p) promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP-FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation. In agreement with previous reports, GFP-Faa2p was found to localize to peroxisomes.
Abstract: The yeast vacuole functions both as a degradative organelle and as a storage depot for small molecules and ions. Vacuoles are dynamic reticular structures that appear to alternately fuse and fragment as a function of growth stage and environment. Vac8p, an armadillo repeat-containing protein, has previously been shown to function both in vacuolar inheritance and in protein targeting from the cytoplasm to the vacuole. Both myristoylation and palmitoylation of Vac8p are required for its efficient localization to the vacuolar membrane (Y.-X. Wang, N. L. Catlett, and L. S. Weisman, J. Cell Biol. 140:1063-1074, 1998). We report that mutants with conditional defects in the rate-limiting enzyme of fatty acid synthesis, acetyl coenzyme A carboxylase (ACC1), display unusually multilobed vacuoles, similar to those observed in vac8 mutant cells. This vacuolar phenotype of acc1 mutant cells was shown biochemically to be accompanied by a reduced acylation of Vac8p which was alleviated by fatty acid supplementation. Consistent with the proposed defect of acc1 mutant cells in acylation of Vac8p, vacuolar membrane localization of Vac8p was impaired upon shifting acc1 mutant cells to nonpermissive condition. The function of Vac8p in protein targeting, on the other hand, was not affected under these conditions. These observations link fatty acid synthesis and availability to direct morphological alterations of an organellar membrane.
Abstract: The yeast translational activator protein Cbs2p is imported into mitochondria without obvious proteolytic processing. To test the importance of amino-terminal amino acids for mitochondrial targeting we fused varying portions of the N-terminus with green fluorescent protein and examined the intracellular distribution of the reporter protein. We show that the 25 N-terminal amino acids are sufficient to direct the majority of the fusion protein into mitochondria. Cbs2p derivatives lacking 9 to 35 amino acids from the N-terminus fail to complement the respiratory deficiency of a deltacbs2 strain, but are still imported into mitochondria. Therefore Cbs2p contains at least one independent mitochondrial targeting information in addition to the N-terminal signal. We further analyzed the effect of over-expression of Cbs2p on mitochondrial function. Elevated concentrations of Cbs2p lead to slightly impaired mitochondrial gene expression, probably as the result of the formation of inactive Cbs2p aggregates.
Abstract: The yeast ERG4 gene encodes sterol C-24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol. Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta-5,7,22,24(28)-tetraen-3beta-ol. An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4-nitroquinoline, fluconazole, and sodium dodecyl sulfate. Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi-destabilizing drug brefeldin A. Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum. This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein. We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations.
Abstract: The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.
Abstract: The yeast Saccharomyces cerevisiae is a very powerful system for cell biological research. Recent advances in electronic light microscopy together with the application of green fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights into structural organization and dynamics of cells as small as yeast. Methods for staining yeast for microscopic inspection and for introducing tags for localization studies of proteins in living or fixed cells are summarized. Electronic light microscopy, video/deconvolution methods, and confocal laser scanning microscopy as novel tools for structural analyses, and their practical applications in yeast, are discussed.
Abstract: The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.
Abstract: Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.
Abstract: The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.
Abstract: In a screen for mutants that display synthetic lethal interaction with hpr1Delta, a hyperrecombination mutant of Saccharomyces cerevisiae, we have isolated a novel cold-sensitive allele of the acetyl coenzyme A (CoA) carboxylase gene, acc1(cs), encoding the rate-limiting enzyme of fatty acid synthesis. The synthetic lethal phenotype of the acc1(cs) hpr1Delta double mutant was only partially complemented by exogenous fatty acids. hpr1Delta was also synthetically lethal with a previously isolated, temperature-sensitive allele of ACC1, mtr7 (mRNA transport), indicating that the lethality of the acc1(cs) hpr1Delta double mutant was not allele specific. The basis for the interaction between conditional acc1 alleles and hpr1Delta was investigated in more detail. In the hpr1Delta mutant background, acetyl-CoA carboxylase enzyme activity was reduced about 15-fold and steady-state levels of biotinylated Acc1p and ACC1 mRNA were reduced 2-fold. The reduced Acc1p activity in hpr1Delta cells, however, did not result in an altered lipid or fatty acid composition of the mutant membranes but rendered cells hypersensitive to soraphen A, an inhibitor of Acc1p. Similar to mtr7, hpr1Delta and acc1(cs) mutant cells displayed a defect in nuclear export of polyadenylated RNA. Oversized transcripts were detected in hpr1Delta, and rRNA processing was disturbed, but pre-mRNA splicing appeared wild type. Surprisingly, the transport defect of hpr1Delta and acc1(cs) mutant cells was accompanied by an altered ring-shaped structure of the nucleolus. These observations suggest that the basis for the synthetic lethal interaction between hpr1Delta and acc1 may lie in a functional overlap of the two mutations in nuclear poly(A)+ RNA production and export that results in an altered structure of the nucleolus.
Abstract: Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.
Abstract: Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.
Abstract: Acetate non-utilizing strains harbouring trans-dominant mutations in the GPR1 gene (GPR1(d)) of the dimorphic yeast Yarrowia lipolytica have been selected and characterized. These mutants are highly sensitive to low concentrations of acetic acid and ethanol, even in presence of glucose. The toxic effect of acetic acid is pH-dependent and has the strongest effect at low pH. In contrast, the action of ethanol is pH-independent. One GPR1(d) mutant has been detected that was highly sensitive to acetic acid but could still grow on ethanol, which indicates putative differences in the function of the GPR1 gene product in the sensitivity to acetic acid and ethanol. The GPR1(d) mutants exhibit a complex pleiotropic phenotype. The mutations cause changed colony morphology as well as dimorphism of cells, and induce early cell death during growth on glucose, even without the presence of dicarbon compounds. Composition of intracellular membranes, as well as morphology of vacuole and mitochondria, were strongly changed. Back-crosses with wild-type strains and analysis of recombinant strains have shown that the expression of the pleiotropic phenotype depends on the site of mutation in the GPR1 gene, as well as on the genetic background of the strain harbouring the responsive mutation. Our data suggest that Gpr1p is involved in a general response of cells to the toxic action of dicarbon compounds like acetic acid and ethanol.
Abstract: An Autonomously Replicating Sequence element adjacent to the RIB1 gene encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identified by transformation experiments. Detailed sequence analysis unveiled two potential ARS elements located 5' and 3' of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like sequence 3' to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 10(4)-10(5) transformants/microg of DNA to a pUC19-derived plasmid in P. guilliermondii. The PgARS element also conferred autonomous replication to hybrid plasmids in this host. Based on this element a series of Escherichia coli shuttle vectors for efficient transformation of the flavinogenic yeast P. guilliermondii was developed.
Abstract: Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.
Abstract: The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3',4'-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lyso-analogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent.
Abstract: The construction of six deletion mutants of Saccharomyces cerevisiae and their basic phenotypic characterization are described. Open reading frames YDL148c, YDL109c, YDL021w, YDL019c, YDL018c and YDL015c from the left arm of chromosome IV were deleted using a polymerase chain reaction (PCR)-based disruption technique, introducing the kanMX4 resistance marker into the respective genes. Gene replacement cassettes (pYORCs) for use in other strain backgrounds were cloned by PCR using DNA templates from haploid or diploid deletion mutants, and inserted into episomal plasmids. Cognate clones of all six ORFs were obtained by gap repair. Deletions were carried out in diploid cells and, after sporulation, yielded four viable spores for clones disrupted in YDL109c, YDL021w, YDL019c and YDL018c. Spores harbouring disruptions in ORFs YDL148c and YDL015c germinated but underwent only a few divisions before ceasing growth, suggesting that the respective genes are essential for vegetative growth on YPD complete media. The other deletion mutants grew like wild-type at different temperatures and on different carbon sources. A brief computational analysis of the six ORFs studied in this work is presented.
Abstract: Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem. 270, 29836-29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.
Abstract: Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non-host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance-inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS-acetate. Using Pir7b-specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.
Abstract: Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.
Abstract: The effects of 4-hydroxy-2-nonenal (HNE) on the cell division cycle were investigated in the yeast Saccharomyces cerevisiae. A short treatment with HNE at a concentration in the range of the IC50 value in S. cerevisiae SP-4 cells induced a significant increase in the proportion of G0/G1 cells at the expense of S-phase cells. A similar delay in cell cycle progression upon treatment with HNE has recently been shown for HL-60 neoplastic cells. Long-term exposure in a synchronized yeast culture resulted in a pronounced dose-dependent block between G0G1- and S-phase, probably at or close to a point in the cell cycle that has been designated as "START." Incorporation of radioactively labeled precursors of macromolecules revealed that DNA synthesis was most susceptible to HNE in comparison to RNA and protein synthesis. Production of glutathione appeared to be required for the continuation of the cell cycle. HNE-treated yeast cells reentered the cell cycle when their glutathione content exceeded about twice the level of control cells. The release from the cell division cycle delay was followed by an enhanced growth to an extent that HNE-treated cells exceeded the number of control cells. These results indicate that HNE causes a biphasic modulation of cell proliferation. It was concluded that this effect was conserved during evolution from yeast to mammalian cells, emphasizing once more the usefulness of this unicellular organism as a model system for the investigation of the effects of free radical-derived products on the proliferation of eukaryotes.
Abstract: Acetyl-CoA carboxylase (ACC1) catalyzes the first and rate limiting step of de novo fatty acid synthesis. Defects in Acc1p were recently correlated with an altered structure/function of the nuclear envelope in yeast. The subcellular distribution of the enzyme was determined in wild-type and mutant cells by cell fractionation and confocal immunofluorescence microscopy. Even though fatty acid synthesis is generally considered to be a cytosolic reaction, we found that Acc1p cofractionated with nuclei and the ER (endoplasmic reticulum) marker BiP/Kar2p. Membrane-bound Acc1p was susceptible to proteinase K digestion and was solubilized by mild salt treatment indicating that it is loosely associated with the cytosolic surface of the nuclear ER membrane. Consistent with these observations, immunofluorescence analysis revealed that Acc1p was distributed in a gradient within the cytoplasm that had its highest concentration around the ER. Possible association of Acc1p with the nuclear pore complexes (NPCs) was investigated in strains that display NPC clustering. Results of these experiments suggest that Acc1p localization is independent of NPC distribution. We propose that association of Acc1p with the cytoplasmic surface of the ER membrane is physiologically relevant to "channel" the enzymatic product of Acc1p, malonyl-CoA, to a putative ER-localized fatty acid chain elongase complex.
Abstract: (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.
Abstract: (S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C--C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the alpha/beta hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis.
Abstract: The mic2 mutation dominantly blocks formation of mannosyl-diinositolphosphorylceramide, the most abundant sphingolipid of the yeast, Saccharomyces cerevisiae. Interestingly, lack of mannosyl-diinositolphosphorylceramide is not lethal but is compensated for by increased amounts of inositolphosphorylceramide and mannosyl-inositolphosphorylceramide in the plasma membrane and Golgi of the mutant. The level of negatively charged phospholipids in the plasma membrane of the mic2 strain is markedly reduced; the sterol composition is not altered. In spite of dramatic changes of its lipid composition the mutant grows like wild type on complex and minimal media, under osmotic stress conditions, at low pH, and in the presence of high ionic strength. While sensitivity to several drugs is not altered, the mic2 mutant strain becomes resistant to the polyene antibiotic nystatin.
Abstract: The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE). GSH was entirely depleted within a 2 h incubation with 250 microM HNE. After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level. In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells. We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions. Furthermore, we have discovered a new indication for the stress marker trehalose in S. cerevisiae.
Abstract: A membrane-bound phospholipase D (PLD) from Saccharomyces cerevisiae was solubilized from mitochondrial and plasma membranes and partially purified. The enzyme has an apparent molecular weight of approximately 60 kDa, is strictly Ca(2+)-dependent and preferentially hydrolyses phosphatidylserine and phosphatidylethanolamine. Enzyme activity is significantly increased in membranes from cells grown on a non-fermentable carbon source. The Ca(2+)-dependent PLD is distinct from PLD encoded by the SPO14IPLD1 gene. The 195 kDa SPO14IPLD1 gene product is specific for PtdCho, Ca(2+)-independent and is activated by PIP2. Furthermore, Pld1p has transphosphatidylation activity in the presence of ethanol and thus resembles the prototypic PLD activity found in mammalian cells and plants. In contrast, the Ca(2+)-dependent PLD described here is not affected by PIP2 and does not catalyze transphosphatidylation. Thus, the Ca(2+)-dependent PLD characterized in this study appears to be a member of a novel family of phospholipases D.
Abstract: The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.
Abstract: Cells of the pel1 mutant of Saccharomyces cerevisiae were found to contain an extremely low content of cardiolipin, a decreased level of phosphatidylcholine and an increased level of phosphatidylinositol. Disruption of the PEL1 gene in cells containing a null mutation in the CHO1 gene was lethal. Despite its putative functional homology with CHO1, the overexpression of the PEL1 gene in the cho1 null mutant did not restore the wild-type properties of the transformed cells and failed to stimulate the incorporation of L-[3-3H]serine into total lipids of the intact yeast cells.
Abstract: The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.
Abstract: Most of the enzymes and genes required for lipid biosynthesis and degradation in the budding yeast Saccharomyces cerevisiae have now been identified and the global mechanisms that regulate their activity are being established. Synthesis of phospholipids is restricted to specific subcellular compartments, and the lipids migrate from their site of formation to their final destination. In addition to synthesis, remodelling and degradation of phospholipids controls the content of the lipid portion of cellular membranes, while highly specific phospholipases catalyse the release of lipid-based second messengers. In this review, we describe the current understanding of the organization and regulation of phospholipid metabolism in yeast, and discuss the mechanisms that have been proposed for intracellular lipid transport.
Abstract: The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38,711 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type II from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiae. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits.
Abstract: By screening a collection of Saccharomyces cerevisiae mutants temperature sensitive for growth on a nonfermentable carbon source, we have isolated a gene (termed MAS37) which encodes a novel receptor for protein import into mitochondria. Mas37p is a 37-kD outer membrane protein with two putative membrane-spanning regions. Inactivation of the MAS37 gene renders cells temperature-sensitive for respiration-driven growth, inhibits import of precursors into isolated mitochondria, and is synthetically lethal with a deletion of one of the genes encoding the import receptors Mas70p or Mas20p. Inactivation of Mas37p with specific antibodies inhibits import of different precursors to different extents; the precursor specificity of Mas37p resembles that of the previously described import receptor Mas70p. Mas70p and Mas37p form a 1:1 complex in detergent extracts of mitochondria and overexpression of one protein enhances that of the other. We suggest that the Mas37p/Mas70p heterodimer functions as a receptor for protein import into yeast mitochondria and that the mitochondrial receptor system consists of hetero-oligomeric subcomplexes with distinct binding activities, but overlapping precursor specificities.
Abstract: Several enzymes with lysophospholipase/phospholipase B activity have been described from the budding yeast Saccharomyces cerevisiae. In vitro, these enzymes are capable of hydrolyzing all phospholipids that can be extracted from yeast cells. Two forms of the enzyme have been isolated from plasma membranes and a third from culture supernatants and the periplasmic space, but their biological roles have not been determined. These highly glycosylated enzymes were reported to have very similar catalytic properties but differed with respect to apparent molecular weight. We isolated a gene from S. cerevisiae, encoding a protein predicted to share 45% amino acid sequence identity with phospholipase B from Penicillium notatum. This yeast gene, designated PLB1, was mapped to the left arm of chromosome VIII. No residual lysophospholipase/phospholipase B activity was detected upon assay of extracts or culture supernatants of a plb1 delta mutant. Thus, either the PLB1 gene encodes all of the previously detected isoforms of phospholipase B or its gene product is required for their expression or activation. Deletion of PLB1 did not result in any apparent phenotypic defect, suggesting either that we failed to identify the growth conditions that would betray such a defect or that Plb1p is functionally redundant with another protein, whose activity has gone undetected. A plb1 delta mutant released wild-type levels of the soluble phosphatidylinositol metabolite glycerophosphoinositol into the growth medium but released greatly reduced levels of the corresponding phosphatidylcholine and phosphatidylethanolamine metabolites. These results indicate that PLB1 is principally responsible for the production of the deacylation products of phosphatidylcholine and phosphatidylethanolamine but not phosphatidylinositol.
Abstract: The isolation of the dep1 mutant of Saccharomyces cerevisiae is reported. The mutant was identified by its disability to regulate expression of structural genes involved in phospholipid biosynthesis, INO1, CHO1 and OPI3, in response to supplementation with soluble lipid precursors. Expression of the INO1, CHO1 and OPI3 genes was not fully derepressed in the absence of soluble lipid precursors, inositol and choline in the dep1 mutant, as compared to wild type. The mutant also exhibited incomplete repression of these same genes in the presence of inositol and choline. Repression by phosphate of the PHO5 gene was reduced in the mutant, as was derepression of this gene in the absence of phosphate. In addition, we show that expression of INO1 and OPI3 structural genes is strongly dependent on the growth phase both in wild-type and dep1 mutant strains. However, in the mutant, elevated basal steady-state mRNA levels were reached in the late stationary growth phase, independent of supplementation conditions. The dep1 mutation represents a new complementation group with respect to phospholipid synthesis and was mapped to a position of about 12 cM distal from the centromere on the left arm of chromosome I. Deficiencies in transcription activation and repression of metabolically unrelated genes, as well as reduced mating efficiency and lack of sporulation of homozygous diploid dep1/dep1 mutants indicate a pleiotropic regulatory function of the DEP1 gene product. Thus, Dep1p appears to be a new member of a class of transcriptional modulators, including Rpd1p/Sin3p/Ume4p/Sdi1p/Gam3p, Rpd3p, Spt10p and Spt21p.
Abstract: The use of beta-glucuronidase (beta-GUS) as a reporter and sensitive detection system for Yarrowia lipolytica was studied. The Escherichia coli gusA gene was expressed under control of the homologous LEU2 promoter in a transcriptional fusion. An NcoI restriction site was introduced at the translational start-ATG, conserving the most favorable context for initiation of translation. The chimeric LEU2'-gusA gene was integrated into the LEU2 locus by homologous recombination. The beta-GUS assay was very sensitive and highly reproducible, using the cytosolic fraction or a total cell extract as the source of enzyme. In a leucine-free medium, beta-GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, beta-GUS activity was reduced about three-fold, but doubled during logarithmic growth. No intrinsic beta-GUS activity was detectable in untransformed Y. lipolytica and no effect of beta-GUS expression on growth was observed. beta-GUS-producing Y. lipolytica cells could be directly detected on media plates containing X-gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide).
Abstract: We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.
Abstract: By functional complementation of the auxotrophic requirements for choline of a cdg1, cho2 double-mutant, by transformation with a genomic DNA library in a high copy number plasmid, two different types of complementing DNA inserts were identified. One type of insert was earlier shown to represent the CHO2 structural gene. In this report we describe the molecular and biochemical chemical characterization of the second type of complementing activity. The transcript encoded by the cloned gene was about 1000-nt in length and was regulated in response to the soluble phospholipid precursors, inositol and choline. A gene disruption resulted in no obvious growth phenotype at 23 degrees C or 30 degrees C, but in a lack of growth at 37 degrees C in the presence of monomethylethanolamine. Null-mutants exhibited an inositol-secretion phenotype, indicative of mutations in the lipid biosynthetic pathway. Complementation analysis, biochemical analysis of the phospholipid methylation pathway in vivo, and comparison of the restriction pattern of the cloned gene to published sequences, unequivocally identified the cloned gene as the OPI3 gene, encoding phospholipid-N-methyltransferase in yeast. When present in multiple copies the OPI3 gene efficiently suppresses the phospholipid methylation defect of a cho2 mutation. As a result of impaired synthesis of phosphatidylcholine, the INO1-deregulation phenotype is abolished in cho2 mutants transformed with the OPI3 gene on a high copy number plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: A 34 base-pair (bp) fragment spanning sequences -154 to -120 of the promoter of the CHO1 gene (structural gene for phosphatidylserine synthase) from the yeast Saccharomyces cerevisiae has been shown to place transcription of a promoter-less Escherichia coli lacZ gene under control of the phospholipid precursors inositol and choline. Furthermore, in deletion experiments the CHO1 UASINO was localized to sequences between -151 and -123 of the CHO1 promoter. A nine bp sequence was identified in the promoter region of the CHO1 gene that shares an eight out of nine bp match with a sequence (consensus 5' ATGTGAAAT 3') that is repeated a total of 23 times upstream from several coregulated phospholipid biosynthetic genes. This sequence is contained within the -151 to -123 region to which the CHO1 UAS has been localized. The nine bp repeated element is believed to be involved in the control of phospholipid biosynthetic gene transcription in response to changing levels of inositol and choline in the growth medium. This control has been shown to require activities encoded by the products of the three regulatory genes: INO2, INO4, and OPI1. A mutation in any of these regulatory genes results in aberrant CHO1-lacZ gene regulation, and affects regulation of the construct containing the 34 bp (-154 to -120) CHO1 fragment demonstrating that the regulatory signal generated by these genes interacts with the 5' end of the CHO1 gene.
Abstract: Regulation of the activity of the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) was measured in vitro by using membrane preparations from wild-type and mutant strains of Saccharomyces cerevisiae. PSD specific activity was not affected by carbon source, and on all carbon sources, the highest specific activity was observed in cells entering the stationary phase of growth. However, PSD activity was found to be regulated in response to soluble precursors of phospholipid biosynthesis. PSD specific activity was reduced to about 63% of the level observed in unsupplemented wild-type cells when the cells were grown in the presence of 75 microM inositol. The presence of 1 mM choline alone had no repressing effect, but the presence of 1 mM choline and 75 microM inositol together led to further repression to a level of about 28% of the derepressed activity. Regulatory mutations known to affect regulation or expression of genes encoding phospholipid-synthesizing enzymes also affected PSD specific activity. opi1 mutants, which are constitutive for a number of phospholipid-biosynthetic enzymes, were found to have high, constitutive levels of PSD. Likewise, in ino2 or ino4 regulatory mutants, PSD activity was found to be at the fully repressed level regardless of growth condition. Regulation of PSD activity was also affected in several structural-gene mutants under conditions of impaired phosphatidylcholine biosynthesis. Together, these data strongly suggest that PSD expression is controlled by the mechanism of general control of phospholipid biosynthesis that regulates many enzymes of phospholipid biosynthesis.
Abstract: Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.
Abstract: The product of the yeast CHO 1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1.2 kb fragment containing the regulatory and structural components of the CHO 1 gene was sequenced. Transcription initiation in wild-type cells was found to occur between -1 and -15 relative to the first ATG of a large open reading frame capable of encoding a 30,804 molecular weight protein. This translation initiation site was active in vivo and in vitro in a heterologous system. In both cases it supported production of a protein of approximately 30,000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases downstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22,400 molecular weight. A subclone, lacking the 5' regulatory region and the sequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22,000 molecular weight, lacked the 74 N-terminal amino acids and was capable of complementing the choline auxotrophy of a cho 1 null-mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrid-protein containing the 63 N-terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild-type yeast. Thus, the hydrophilic, highly acidic N-terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.
Abstract: The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind 14C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37 degrees C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added 14C-palmitic acid was significantly reduced both at 30 and 37 degrees C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.
Abstract: A yeast cDNA genetic library in a bacteriophage expression vector was screened using an antiserum reacting with fructose 1,6-bisphosphate aldolase from Saccharomyces cerevisiae. Radio-labelled probes of selected immunopositive clones were used for screening of a yeast genomic library. From the genomic clones a yeast/Escherichia coli shuttle plasmid was constructed containing on a 1990-base-pair fragment the entire structural gene FBA1 coding for yeast aldolase. The primary structure of the FBA1 gene was determined. An open reading frame comprises 1077 base pairs coding for a protein of 359 amino acids with a predicted molecular mass of 39,608 Da. As observed for other strongly expressed yeast genes, codon usage is extremely biased. The 810 base pairs at the 5' end and the 90 base pairs at the 3' end of the coding region of the cloned FBA1 gene are sufficient for normal expression and show characteristic elements present in the noncoding sequences of other yeast genes. Aldolase is the major protein in yeast cells transformed with a high-copy-number plasmid containing the FBA1 gene. The aldolase gene was disrupted by insertion of the yeast URA3 gene into the coding region of one FBA1 allele in a homozygous diploid ura3 strain. The haploid offsprings with the defective aldolase allele fba1::URA3 lack aldolase enzymatic activity and fail to grow in media containing as a carbon source metabolites of only one side of the aldolase reaction.
Abstract: In Saccharomyces cerevisiae, the membrane-associated enzyme phosphatidylserine synthase (EC 2.7.8.8) is present in the mitochondria and the endoplasmic reticulum. The enzyme from both membrane fractions reacted with antiserum raised against a hybrid protein expressed from a TRPE-CHO1 fusion gene in Escherichia coli and was absent in a cho1 null mutant, strongly suggesting that both the mitochondrial and microsomal forms of phosphatidylserine synthase are the products of the CHO1 gene. The highest degree of purification of enzymatically active protein was 380- and 420-fold from the mitochondrial and the microsomal compartments, respectively. In both cases, the enzymatically active and immunoreactive material comigrated with a protein band of 30,000 apparent molecular weight. In the absence of protease inhibitors during the preparation of membranes, the enzyme underwent degradation to an enzymatically active protein of 23,000 apparent molecular weight.
Abstract: A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.
Abstract: Yeast cells take up exogenous fatty acids with subsequent rapid incorporation into glycerolipids. beta-Oxidation does not occur in Saccharomyces uvarum and is observed in Saccharomycopsis lipolytica only 2-5 min after addition of radioactively labeled fatty acid. Rates of fatty acid uptake are linear up to 30 s with S. lipolytica and up to 2 min with S. uvarum. The uptake kinetics are consistent with a dual mode of transport, comprising a saturable component with KT values in the range 10(-5)-10(-6) M, and apparently simple diffusion that predominates at high substrate concentrations. Kinetics of fatty acid permeation are independent of metabolic energy and membrane potential. At least two fatty acid carrier systems exist in both S. lipolytica and S. uvarum, one being specific for fatty acids with 12 and 14 C atoms, respectively, the other for C16 and C18 saturated or unsaturated fatty acids. Octanoic acid and decanoic acid are not taken up by S. lipolytica. Internalization of lauric acid and oleic acid by S. lipolytica cells is preceded by a rapid (less than 5 s) initial uptake which most likely represents irreversible adsorption. This phenomenon was not observed with heat-inactivated S. lipolytica cells or with viable S. uvarum. In azide-poisoned cells of S. lipolytica an up to 20-fold accumulation of unesterified fatty acid was observed within 30 s after the addition of substrate.
