Abstract: The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses.

Abstract: Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed by two distinct subunits, alpha (catalytic), and beta (regulatory), which combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss-of-function of CK2 on plant development we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open-reading-frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not due to disruption of light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion with compelling effects on Arabidopsis development.

Abstract: Failure to use glucose as carbon source results in transcriptional activation of numerous genes whose expression is otherwise repressed. HXT2 encodes a yeast high-affinity glucose transporter that is only expressed under conditions of glucose limitation. We show that HXT2 is rapidly and potently induced by environmental alkalinization and this requires both the Snf1 and the calcineurin pathways. Regulation by calcineurin is mediated by the transcription factor Crz1, which rapidly translocates to the nucleus upon high pH stress, and acts through a previously unnoticed Crz1-binding element (CDRE) in the HXT2 promoter (-507 GGGGCTG -501). We demonstrate that, in addition to HXT2, many other genes required for adaptation to glucose shortage, such as HXT7, MDH2 or ALD4, transcriptionally respond to calcium and high pH signaling through binding of Crz1 to their promoters. Therefore, calcineurin-dependent transcriptional regulation appears to be a common feature for many genes encoding carbohydrate-metabolizing enzymes. Remarkably, extracellular calcium allows growth of a snf1 mutant on low glucose in a calcineurin/Crz1 dependent-manner, indicating that activation of calcineurin is sufficient to override a major deficiency in the glucose-repression pathway. We propose that alkalinization of the medium results in impaired glucose utilization and that activation of certain glucose-metabolizing genes by calcineurin contributes to yeast survival under this stress situation.

Abstract: Regulatable gene expression is a powerful genetic tool for analyzing the function of a given gene product. The use of tetracycline-regulatable promoters in yeast represents a substantial improvement over previously described methods for gene regulation. Here we show how this approach can be used to analyze the biological role of serine/threonine phosphatase catalytic or putative regulatory subunits by constructing chromosomal or plasmid-borne conditional mutants. This is particularly useful given the large variety of important biological processes performed by these of enzymes, often necessaries for cell survival, which makes in some cases infeasible the generation of null mutants.

Abstract: We have recently characterized Ypi1 as an inhibitory subunit of yeast Glc7 PP1 protein phosphatase. In this work we demonstrate that Ypi1 forms a complex with Glc7 and Sds22, another Glc7 regulatory subunit that targets the phosphatase to substrates involved in cell cycle control. Interestingly, the combination of equimolar amounts of Ypi1 and Sds22 leads to an almost full inhibition of Glc7 activity. Because YPI1 is an essential gene, we have constructed conditional mutants that demonstrate that depletion of Ypi1 leads to alteration of nuclear localization of Glc7 and cell growth arrest in mid-mitosis with aberrant mitotic spindle. These phenotypes mimic those produced upon inactivation of Sds22. The fact that progressive depletion of either Ypi1 or Sds22 resulted in similar physiological phenotypes and that both proteins inhibit the phosphatase activity of Glc7 strongly suggest a common role of these two proteins in regulating Glc7 nuclear localization and function.

Abstract: The efficiency of stop codons read-through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3-Ppz1 protein complex in regulation of read-through efficiency and manifestation of non-Mendelian anti-suppressor determinant [ISP(+)]. Over-expression of HAL3 in [ISP(+)] strain causes nonsense suppression, whereas its inactivation displays as anti-suppression of sup35 mutation in [isp(-)] strain. [ISP(+)] strains carrying hal3Delta deletion cannot be cured from [ISP(+)] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over-expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Balpha elongation factor activity. Importantly, [ISP(+)] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP(+)] strains have less Hal3p than their [isp(-)] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP(+)] status of yeast cell is suggested.

Abstract: Zinc is an essential metal that, when in excess, can be deleterious to the cell. Therefore, homeostatic mechanisms for this cation must be finely tuned. To better understand the response of yeast in front of an excess of zinc, we screened a systematic deletion mutant library for altered growth in the presence of 6 mM zinc. Eighty-nine mutants exhibited increased zinc sensitivity, including many genes involved in vacuolar assembling and biogenesis. Interestingly, a mutant lacking the Aft1 transcription factor, required for the transcriptional response to iron starvation, was found to be highly sensitive to zinc. Genome-wide transcriptional profiling revealed that exposure to 5 mM ZnCl(2) results in rapid increase in the expression of numerous chaperones required for proper protein folding or targeting to vacuole and mitochondria, as well as genes involved in stress response (mainly oxidative), sulphur metabolism and some components of the iron regulon. The effect of the lack of Aft1 both in the absence and in the presence of zinc overload was also investigated. Exposure to high zinc generated reactive oxygen species and markedly decreased glutathione content. Interestingly, zinc excess results in decreased intracellular iron content and aconitase and cytochrome c activities in stationary-phase cultures. These findings suggest that high zinc levels may alter the assembly and/or function of iron-sulphur-containing proteins, as well as the biosynthesis of haem groups, thus establishing a link between zinc, iron and sulphur metabolism.

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Abstract: The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na(+)-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance.

Abstract: Protein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild-type cells and that this is most probably the result of increased expression of the ENA1 Na(+)-ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium-sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1-4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li(+) and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.

Abstract: Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.

Abstract: Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na(+)-ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5.-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5. end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.

Abstract: Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.

Abstract: The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.

Abstract: Exposure of the yeast Saccharomyces cerevisiae to an alkaline environment represents a stress situation that negatively affects growth and results in an adaptive transcriptional response. We screened a collection of 4825 haploid deletion mutants for their ability to grow at mild alkaline pH, and we identified 118 genes, involved in numerous cellular functions, whose absence results in reduced growth. The list includes several key genes in copper and iron homeostasis, such as CCC2, RCS1, FET3, LYS7, and CTR1. In contrast, a screen of high-copy number plasmid libraries for clones able to increase tolerance to alkaline pH revealed only two genes: FET4 (encoding a low affinity transporter for copper, iron, and zinc) and CTR1 (encoding a high affinity copper transporter). The beneficial effect of overexpression of CTR1 requires a functional high affinity iron transport system, as it was abolished by deletion of FET3, a component of the high affinity transport system, or CCC2, which is required for assembly of the transport system. The growth-promoting effect of FET4 was not modified in these mutants. These results suggest that the observed tolerance to alkaline pH is because of improved iron uptake and indicate that both iron and copper are limiting factors for growth under alkaline pH conditions. Addition to the medium of micromolar concentrations of copper or iron ions drastically improved growth at high pH. Supplementation with iron improved somewhat the tolerance of a fet3 strain but was ineffective in a ctr1 mutant, suggesting the existence of additional copper-requiring functions important for tolerance to an alkaline environment.

Abstract: Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.

Abstract: The yeast Saccharomyces cerevisiae has been widely used for the implementation of DNA chip technologies. For this reason and due to the extensive use of this organism for basic and applied studies, yeast DNA chips are being used by many laboratories for expression or genomic analyses. While membrane arrays (macroarrays) offer several advantages, for many laboratories they are not affordable. Here we report that a cluster of four Spanish molecular-biology yeast laboratories, with relatively small budgets, have developed a complete set of probes for the genome of S. cerevisiae. These have been used to produce a new type of macroarray on a nylon surface. The macroarrays have been evaluated and protocols for their use have been optimized.

Abstract: Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.

Abstract: The Ppz protein phosphatases have been recently shown to negatively regulate the major potassium transport system in the yeast Saccharomyces cerevisiae, encoded by the TRK1 and TRK2 genes. We have found that, in the absence of the Trk system, Ppz mutants require abnormally high concentrations of potassium to proliferate. This can be explained by the observation that trk1 trk2 ppz1 or trk1 trk2 ppz1 ppz2 strains display a very poor rubidium uptake, with markedly increased Km values. These cells are very sensitive to the presence of several toxic cations in the medium, such as hygromicyn B or spermine, but not to lithium or sodium cations. At limiting potassium concentrations, addition of EGTA to the medium improves growth of these mutants. Therefore, our results indicate that, in addition to their role in regulating Trk potassium transporters, Ppz phosphatases (essentially Ppz1), positively affect the residual low affinity potassium transport mechanisms in yeast. These findings may provide a new way to elucidate the molecular nature of the low affinity potassium uptake system in yeast as well as a useful model to analyze the function of plant or mammalian potassium channels through heterologous expression in yeast.

Abstract: Inactivation of HAL3 in the absence of SIT4 function leads to cell cycle arrest at the G(1)-S transition. To identify genes potentially involved in the control of this phase of the cell cycle, a screening for multicopy suppressors of a conditional sit4 hal3 mutant (strain JC002) has been developed. The screening yielded several genes known to perform key roles in cell cycle events, such as CLN3, BCK2 or SWI4, thus proving its usefulness as a tool for this type of studies. In addition, this approach allowed the identification of additional genes, most of them not previously related to the regulation of G(1)-S transition or even without known function (named here as VHS1-3, for viable in a hal3 sit4 background). Several of these gene products are involved in phospho-dephosphorylation processes, including members of the protein phosphatase 2A and protein phosphatases 2C families, as well as components of the Hal5 protein kinase family. The ability of different genes to suppress sit4 phenotypes (such as temperature sensitivity and growth on non-fermentable carbon sources) or to mimic the functions of Hal3 was evaluated. The possible relationship between the known functions of these suppressor genes and the progress through the G(1)-S transition is discussed.

Abstract: The yeast Nha1 Na(+),K(+)/H(+) antiporter may play an important role in regulation of cell cycle, as high-copy expression of the NHA1 gene is able to rescue the blockage at the G(1)/S transition of cells lacking Sit4 protein phosphatase and Hal3 activities. Interestingly, this function was independent of the role of the antiporter in improving tolerance to sodium cations, it required the integrity of a relatively large region (from residues 800 to 948) of its carboxy-terminal moiety, and was not performed by the fission yeast homolog antiporter Sod2, which lacks a carboxy-terminal tail. Here we show that a hybrid protein composed of the Sod2 antiporter fused to the carboxy-terminal half of Nha1 strongly increased sodium tolerance, but did not allow growth at high potassium nor did rescue growth of the sit4 hal3 conditional mutant strain. Deletion of Nha1 residues from 800 to 849, 900 to 925 or 926 to 954 abolished the function of Nha1 in cell cycle without affecting sodium tolerance. A screening for loss-of-function mutations at the 775-980 carboxy-terminal tail of Nha1 has revealed a number of residues required for function in cell cycle, most of them clustering in two regions, from residues 869 to 876 (cluster A) and 918 to 927 (cluster B). The later is rather conserved in other related antiporters, while the former is not.

Abstract: The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.

Abstract: Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na(+)-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at -751 to -667, containing a calcineurin-dependent response element (CDRE), and one downstream region (-573 to -490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants.

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Abstract: During the last decade several novel yeast genes encoding proteins related to the PPP family of Ser/Thr protein phosphatases have been discovered and their functional characterization initiated. Most of these novel phosphatases display intriguing structural features and/or are involved in a number of important functions, such as cell cycle regulation, protein synthesis and maintenance of cellular integrity. While in some cases these genes appear to be restricted to fungi, in others similar proteins can be found in higher eukaryotes. This review will summarize the latest advances in our understanding about how these phosphatases are regulated and fulfil their functions in the yeast cell.

Abstract: Carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) I, which facilitate the transport of medium- and long-chain fatty acids through the peroxisomal and mitochondrial membranes, are physiologically inhibited by malonyl-CoA. Using an "in silico" macromolecular docking approach, we built a model in which malonyl-CoA could be attached near the catalytic core. This disrupts the positioning of the acyl-CoA substrate in the channel in the model reported for both proteins (Morillas, M., Gómez-Puertas, P., Roca, R., Serra, D., Asins, G., Valencia, A., and Hegardt, F. G. (2001) J. Biol. Chem. 276, 45001-45008). The putative malonyl-CoA domain contained His(340), implicated together with His(131) in COT malonyl-CoA sensitivity (Morillas, M., Clotet, J., Rubi, B., Serra, D., Asins, G., Ariño, J., and Hegardt F. G. (2000) FEBS Lett. 466, 183-186). When we mutated COT His(131) the IC(50) increased, and malonyl-CoA competed with the substrate decanoyl-CoA. Mutation of COT Ala(332), present in the domain 8 amino acids away from His(340), decreased the malonyl-CoA sensitivity of COT. The homologous histidine and alanine residues of L-CPT I, His(277), His(483), and Ala(478) were also mutated, which decreased malonyl-CoA sensitivity. Natural mutation of Pro(479), which is also located in the malonyl-CoA predicted site, to Leu in a patient with human L-CPT I hereditary deficiency, modified malonyl-CoA sensitivity. We conclude that this malonyl-CoA domain is present in both COT and L-CPT I proteins and might be the site at which malonyl-CoA interacts with the substrate acyl-CoA. Other malonyl-CoA non-inhibitable members of the family, CPT II and carnitine acetyltransferase, do not contain this domain.

Abstract: The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression. We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport. First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters. Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake. Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake. Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH-regulated genes and facilitating alpha-factor recovery). In addition to providing a coherent explanation for all Ppz-dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint.

Abstract: The inclusion of genetically modified (GM) foods in the human diet has caused considerable debate. There is concern that the transfer of plant-derived transgenes to the resident intestinal microflora could have safety implications. For these gene transfer events to occur, the nucleic acid would need to survive passage through the gastrointestinal tract. The aim of the present study was to evaluate the rate at which transgenes, contained within GM soya and maize, are degraded in gastric and small bowel simulations. The data showed that 80 % of the transgene in naked GM soya DNA was degraded in the gastric simulations, while no degradation of the transgenes contained within GM soya and maize were observed in these acidic conditions. In the small intestinal simulations, transgenes in naked soya DNA were degraded at a similar rate to the material in the soya protein. After incubation for 30 min, the transgenes remaining in soya protein and naked DNA were 52 (sem 13.1) % and 34 (sem 17.5) %, respectively, and at the completion of the experiment (3 h) these values were 5 % and 3 %, respectively. In contrast to the soya transgene, the maize nucleic acid was hydrolysed in the small intestinal simulations in a biphasic process in which approximately 85 % was rapidly degraded, while the rest of the DNA was cleaved at a rate similar to that in the soya material. Guar gum and tannic acid, molecules that are known to inhibit digestive enzymes, did not influence the rate of transgene degradation in soya protein. In contrast guar gum reduced the rate of transgene degradation in naked soya DNA in the initial stages, but the polysaccharide did not influence the amount of nucleic acid remaining at the end of the experiment. Tannic acid reduced the rate of DNA degradation throughout the small bowel simulations, with 21 (sem 5.4) % and 2 (sem 1.8) % of the naked soya DNA remaining in the presence and absence of the phenolic acid, respectively. These data indicate that some transgenes in GM foods may survive passage through the small intestine.

Abstract: The short-time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na+-ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pH-responsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali-induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin-dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH-induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.

Abstract: Maintenance of cellular integrity in Saccharomyces cerevisiae is carried out by the activation of the protein kinase C-mediated mitogen-activated protein kinase (PKC1-MAPK) pathway. Here we report that correct down-regulation of both basal and induced activity of the PKC1-MAPK pathway requires the SIT4 function. Sit4 is a protein phosphatase also required for a proper cell cycle progression. We present evidence demonstrating that the G(1) to S delay in the cell cycle, which occurs as a consequence of the absence of Sit4, is mediated by up-regulation of Pkc1 activity. Sit4 operates downstream of the plasma membrane sensors Mid2, Wsc1, and Wsc2 and upstream of Pkc1. Sit4 affects all known biological functions involving Pkc1, namely Mpk1 activity and cell wall integrity, actin cytoskeleton organization, and ribosomal gene transcription.

Abstract: The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.

Abstract: Exposure of yeast to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which is essential for the induction of gene expression required for cell survival upon osmotic stress. Several genes are regulated in response to osmotic stress by Sko1, a transcriptional repressor of the ATF/CREB family. We show by in vivo coprecipitation and phosphorylation studies that Sko1 and Hog1 interact and that Sko1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylates Sko1 in vitro at multiple sites within the N-terminal region. Phosphorylation of Sko1 disrupts the Sko1-Ssn6-Tup1 repressor complex, and consistently, a mutant allele of Sko1, unphosphorylatable by Hog1, exhibits less derepression than the wild type. Interestingly, Sko1 repressor activity is further enhanced in strains with high protein kinase A (PKA) activity. PKA phosphorylates Sko1 near the bZIP domain and mutation of these sites eliminates modulation of Sko1 responses to high PKA activity. Thus, Sko1 transcriptional repression is controlled directly by the Hog1 MAPK in response to stress, and this effect is further modulated by an independent signaling mechanism through the PKA pathway.

Abstract: Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.

Abstract: A screening for multicopy suppressors of the G(1)/S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na(+),K(+)/H(+) antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na(+)/H(+)-ATPase or the Schizosaccharomyces pombe Sod2 Na(+)/H(+) antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp(266)-Asp(267) selectively abolished Na(+) efflux without modifying K(+) efflux and did not affect the capacity of Nha1 to relieve the G(1) blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800-948 is dispensable for sodium detoxification but necessary for transport of K(+) cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K(+) transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.

Abstract: In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Balpha (formerly eEF1beta). An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells. Pull-down experiments using a glutathione S-transferase (GST)-EF1Balpha fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Balpha fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Balpha in yeast, and this may result in modification of the translational accuracy.

Abstract: Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.

Abstract: The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.

Abstract: Adaptation to changes in extracellular salinity is a critical event for cell survival. Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress. About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4 m NaCl, 10 min). Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress. A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed. Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects. The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions.

Abstract: Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Delta and pbs2Delta cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Delta cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.

Abstract: The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.

Abstract: We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A Bbeta subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.

Abstract: Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.

Abstract: We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ariño, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.

Abstract: Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.

Abstract: Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations. The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.

Abstract: We have disrupted seven open reading frames (ORFs) located in the left arm of chromosome XV of the yeast Saccharomyces cerevisiae. These ORFs, previously discovered by our laboratory during the programme of systematic sequencing of the yeast genome, are YOL152w, YOL151w, YOL149w, YOL130w, YOL128c, YOL125w and YOL124c. In most cases, the short flanking homology (SFH) replacement technique has been used. The mutants were analysed for different phenotypic tests. Disruption of YOL130w (also known as ALR1) produced a lethal phenotype, despite the presence of a highly similar gene in the yeast genome (ALR2/YFL050C). Disruption of YOL149w (also known as DCP1, and encoding an mRNA decapping enzyme) results in lethality in the FY1679 background, although it allows slow growth in the CEN.PK141 background. Disruption of the remaining ORFs did not result in readily detectable phenotypic changes.

Abstract: The open reading frame YGL087c in the budding yeast Saccharomyces cerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin-conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators. Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea. This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c-encoded protein functions in a process related to tolerance to DNA damage. We also show that the mutant phenotype is fully complemented by expression of the human UEV-1A cDNA and we propose that UEV-1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents.

Abstract: Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Abstract: The plant Arabidopsis thaliana contains five isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) that can be grouped into two families, one composed by isoforms PP2A-1, -2 and -5 and the other composed by isoforms PP2A-3 and PP2A-4. An Arabidopsis genomic library was screened and several clones corresponding to genes PP2A-3 and PP2A-4 were isolated and analysed. Both genes span over approximately 4.5kbp and are composed of 11 exons and 10 introns that show identical organization. Their untranslated regions are also highly conserved, suggesting that the two genes derive from a common ancestral gene. However, the position of intron/exon junctions completely differs from that of the human PP2A genes. Two transcription start sites have been found in the PP2A-3 gene, the major one mapping at nucleotide position -188 from the translation start codon, whereas only one is observed in PP2A-4 (-145). Functional gene promoter analysis reveals that elements required for transient expression of PP2A-3 and PP2A-4 on a protoplast system are contained within a region of about 600bp upstream from the transcription start sites. This is the first report on the cloning and characterization of genes encoding catalytic subunits of Ser/Thr protein phosphatases 2A in higher plants.

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Abstract: We have previously shown that the mutation of the Schizosaccharomyces pombe PPZ-like protein phosphatase encoded by the gene pzh1+ results in increased tolerance to sodium and in hypersensitivity to potassium ions. A similar phenotype has also been reported for deletants in the spm1/pmk1 gene, encoding a mitogen-activated protein (MAP) kinase. We have found that the sodium tolerance phenotype of pzh1 deletants is stronger than that of spm1 mutants, and both effects are additive. Therefore, most probably both gene products mediate different pathways on sodium tolerance. In our hands, mutation of the kinase does not alter the tolerance to potassium, but it yields cells more tolerant to magnesium ions. While in budding yeast the mutations are synthetically lethal, fission yeast cells lacking both the phosphatase and the kinase genes are viable. Interestingly, their ability to export H+ to the medium is greatly impaired (although not that of pzh1 or spm1 single mutants). We have observed that, although the amount of the H+-ATPase in the plasma membrane is not altered, the activity of the enzyme is lower than normal and cannot be induced by glucose. These observations suggest that the activity of the H+-ATPase in fission yeast might be regulated by phospho-dephosphorylation mechanisms that might involve the pzh1+ and spm1+ gene products.

Abstract: Chromosome XV was one of the last two chromosomes of Saccharomyces cerevisiae to be discovered. It is the third-largest yeast chromosome after chromosomes XII and IV, and is very similar in size to chromosome VII. It alone represents 9% of the yeast genome (8% if ribosomal DNA is included). When systematic sequencing of chromosome XV was started, 93 genes or markers were identified, and most of them were mapped. However, very little else was known about chromosome XV which, in contrast to shorter chromosomes, had not been the object of comprehensive genetic or molecular analysis. It was therefore decided to start sequencing chromosome XV only in the third phase of the European Yeast Genome Sequencing Programme, after experience was gained on chromosomes III, XI and II. The sequence of chromosome XV has been determined from a set of partly overlapping cosmid clones derived from a unique yeast strain, and physically mapped at 3.3-kilobase resolution before sequencing. As well as numerous new open reading frames (ORFs) and genes encoding tRNA or small RNA molecules, the sequence of 1,091,283 base pairs confirms the high proportion of orphan genes and reveals a number of ancestral and successive duplications with other yeast chromosomes.

Abstract: In the yeast Saccharomyces cerevisiae, Na+ efflux is mediated by the Ena1 ATPase, and the expression of the ENA1 gene is regulated by the Ppz1 and Ppz2 Ser/Thr protein phosphatases. On the contrary, in the fission yeast Schizosaccharomyces pombe, effective output of Na+ is attributed to the H+/Na+ antiporter encoded by the sod2 gene. We have isolated a S. pombe gene (pzh1) that encodes a 515-amino-acid protein that is 78% identical, from residue 193 to the COOH terminus, to the PPZ1 and PPZ2 gene products. Bacterially expressed Pzh1p shows enzymatic characteristics virtually identical to those of recombinant Ppz1p. When expressed in high-copy number from the PPZ1 promoter, the pzh1 ORF rescues the caffeine-induced lytic defect and slightly decreases the high salt tolerance of S. cerevisiae ppz1delta mutants. Disruption of pzh1 yields viable S. pombe cells and has virtually no effect on tolerance to caffeine or osmotic stress, but it renders the cells highly tolerant to Na+ and Li+, and hypersensitive to K+. Although lack of pzh1 results in a 2-3-fold increase in sod2 mRNA, the pzh1 mutation significantly increases salt tolerance in the absence of the sod2 gene, suggesting that the phosphatase also regulates a Sod2-independent mechanism. Therefore, the finding of a PPZ-like protein phosphatase involved in the regulation of salt tolerance in fission yeast reveals unexpected aspects of cation homeostasis in this organism.

Abstract: We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

Abstract: A 13421 bp fragment located near the left telomere of chromosome XV (cosmid pEOA461) has been sequenced. Seven non-overlapping open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB859, AOC184, AOE375, AOX142i, AOE423, AOA476 and AOE433). An additional ORF (AOE131) is found within AOA476. Three of them (AOC184, AOA476 and AOE433) show no remarkable identity with proteins deposited in the data banks. ORF AOB859 is quite similar to a hypothetical yeast protein of similar size located in chromosome VI, particularly within the C-terminal half. AOE375 encodes a new member of the glycogen synthase kinase-3 subfamily of Ser/Thr protein kinases. AOX142i is the gene encoding the previously described ribosomal protein L25. AOE423 codes for a protein virtually identical to the MDH2 malate dehydrogenase isozyme. However, our DNA sequence shows a single one-base insertion upstream of the reported initiating codon. This would produce a larger ORF by extending 46 residues the N-terminus of the protein. The existence of this insertion has been confirmed in three different yeast strains, including FY1679.

Abstract: The DNA sequence of a 12,801 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals eight open reading frames (ORFs) encoding polypeptides larger than 100 residues. ORFs AOE129 and AOAA121 are in opposite strands and they overlap at their 3' ends. AOE397 has similarity with phosphofructokinase genes from other organisms and may code for a second 6-phosphofructo-2-kinase of Saccharomyces cerevisiae. Sequence of AOA471 shows significant similarity with yeast genes coding for glycophospholipid-containing proteins. AOD1341 would code for a 1341 amino acids long protein with a predicted ATP/GTP-binding site and a transmembrane domain.

Abstract: Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.

Abstract: The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northern-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.

Abstract: The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.

Abstract: Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.

Abstract: The sequence of a 13 kbp fragment located in the vicinity of the left telomere of chromosome XV (cosmid pEOA179) has been determined. Seven new open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB629, AOA342, AOC231, AOE555, AOE236, AOA236 and AOE1045). Three of them show no identity with proteins deposited in the data banks. ORF AOB629 (629 amino acids) has some similarity with previously described ferric reductases from Saccharomyces cerevisiae and Schizosaccharomyces pombe. ORF AOA342 encodes a polypeptide reminiscent of dihydroflavonol-4-reductases from a number of plant species. AOE236 displays a high level of identity when compared with peroxisomal membrane proteins previously cloned from the methylotrophic yeast Candida boidinii. Finally, AOE1045 encodes a large protein (1045 residues) with some identity with a hypothetical 147 kDa protein identified during the sequencing of Caenorhabditis elegans chromosome 3.

Abstract: The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

Abstract: We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5' terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5'-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.

Abstract: Incubation of hepatocytes from diabetic rats with glucose results in the translocation of glycogen synthase from soluble fractions to fractions which sediment at 9200 g. The extent of the translocation correlates positively with the intracellular concentration of glucose 6-phosphate. No difference was found between healthy and diabetic rats in the capacity of glycogen synthase to translocate to pellets in response to an increase in glucose 6-phosphate. In diabetic hepatocytes, glycogen synthase in the supernatant fractions was not activated upon incubation of the cells with glucose, whereas this sugar was able to activate the enzyme found in the fractions that could be pelleted. In the 9200-g pellets, the glycogen synthase activity ratio (-glucose 6-phosphate/+glucose 6-phosphate) from both healthy and diabetic animals correlated with the intracellular glucose 6-phosphate levels. In the supernatants, the glycogen synthase activity ratio from healthy cells also correlated with glucose 6-phosphate levels. In contrast, in diabetic cells the activation state of the soluble enzyme remained essentially unchanged despite the accumulation of glucose 6-phosphate.

Abstract: Degenerate oligonucleotides were used to selectively amplify yeast genomic sequences related to Ser/Thr protein phosphatases. Among the sequences obtained, clone ST4-2 was found to code for a novel sequence related to previously known phosphatases. A size-selected yeast genomic library was constructed and screened using clone ST4-2 as probe, and one positive clone, named PPG, was isolated. DNA sequencing of a 1.8-kilobase pair fragment of this clone revealed an open reading frame of 1104 base pairs which codes for a 368-amino acid protein. On the basis of its amino acid sequence, the product of gene PPG would be an acidic protein, structurally more related to type 2A than to type 1 or 2B phosphatases, and is characterized by an extension of about 50 amino acids at the carboxyl terminus. The gene, which is located in chromosome XIV, is expressed as a 1.3-kilobase mRNA and is not essential for growth. Haploid mutants carrying a disrupted copy of the gene were able to grow in glucose as well as in other carbon sources, but they accumulated less glycogen than the wild type strain. However, the state of activation of glycogen synthase was essentially identical in wild type and mutant cells. The finding that, in early exponential phase, mutant cells contain higher levels of glycogen phosphorylase a, in addition to a lower amount of total glycogen synthase activity observed in medium-late exponential phase, could account for the difference found in glycogen accumulation.

Abstract: Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79-80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.

Abstract: In a recent paper [Ariño et al., Plant Mol Biol 21: 475-485 (1993)] we reported the amplification of a DNA fragment (AP-2) from the genome of Arabidopsis thaliana encoding an amino acid sequence corresponding to a Ser/Thr protein phosphatase distantly related to type 2A protein phosphatases. In this paper we report the use of the AP-2 fragment to isolate several cDNA clones from a leaf cDNA library. Two of these (EP124 and EP129) largely overlap and contain the AP-2 sequence, whereas a third clone (EP128) is different although very related in sequence (86% of identity). Clones EP124/EP129 and EP128 were found to encode two highly related polypeptides (93% identity) of 305 residues, showing a very high identity (83%) to the catalytic subunit of protein phosphatase X (PPX) from rabbit. Therefore, they have been named PPX-1 (EP124/EP129) and PPX-2 (EP128). Southern blot analysis of genomic DNA indicates that only these two genes encoding phosphatases closely related to PPX are present in the genome of A. thaliana. Both PPX-1 and PPX-2 are expressed at very low levels in A. thaliana flowers, leaves, stems and roots. The expression levels of four previously identified type 2A phosphatases are higher than those of PPX genes. PP2A-1 appears to be the major mRNA species detected in all the tissues analyzed.

Abstract: We have recently reported the existence in the yeast Saccharomyces cerevisiae of a gene named PPZ1, encoding a novel Ser/Thr phosphatase characterized by a large, Ser-rich amino-terminal extension, and suggested the existence of a related gene product that could have overlapping functions. We have now amplified by polymerase chain reaction techniques a genomic fragment of about 600 bp corresponding to this second gene (PPZ2). This fragment hybridizes to an mRNA of about the same size as the PPZ1 message but the amount of PPZ2 mRNA peaks at the stationary phase, when almost no PPZ1 mRNA is found. The PPZ2 fragment was interrupted in vitro and used to transform diploid heterozygous ppz1 PPZ2 cells. Haploid cells carrying the double mutation ppz1 ppz2 were unable to grow in the presence of 5 mM caffeine. However, the mutants did survive when osmotically stabilized in the presence of 1 M sorbitol. The evidence obtained suggests that PPZ1 and PPZ2 may be structurally and functionally related and points to an involvement of these phosphatases in functions related to the maintenance of cell integrity.

Abstract: DNA fragments containing structural characteristics found in Ser/Thr protein phosphatases were amplified by polymerase chain reaction from yeast genome. Amplification was carried out by using degenerate oligonucleotides encoding conserved sequences found in type 1, 2A, and 2B phosphatases. A 215-base pair amplification fragment was used to screen a size-selected library, and a positive clone was isolated and sequenced. Nucleotide sequencing revealed a 2076-base pair open reading frame encoding a 692-amino acid protein. The carboxyl half of the protein is structurally related to type 1 phosphatases and virtually identical with the sequence reported as PPZ1, whereas the amino-terminal half of the protein is unrelated to sequences found in other protein phosphatases. This region is very rich in Ser residues and presents a high number of basic amino acids. Therefore, the gene product, on the basis of its unique structure, would represent a novel class of protein phosphatase. The gene, which is located at chromosome XIII, is transcribed as a mRNA of about 2.7 kilobases, and the amount of message has been found to increase 3- to 4-fold during the culture. The product of the gene PPZ1 was identified by immunoblot analysis of cell extracts as a 75-kDa protein, and the amount of immunoreactive protein was increased in cells carrying multiple copies of the gene. Disruption of the gene resulted in viable cells, with no detectable phenotypic change, indicating that the gene is not essential for growth.

Abstract: Incubation of rat hepatocytes with glucose results in a decrease in the amount of glycogen synthase activity found in supernatants obtained after centrifugation of cell homogenates at 9200 g. The enzymic activity was quantitatively recovered in the sediments. This effect of translocation was dose- and time-dependent and correlated with the amount of immunoreactive enzyme determined by immunoblotting in both fractions. Hydrolysis by alpha-amylase of glycogen accumulated upon incubation with the sugar did not affect the translocation pattern. Translocation was also observed when cells were incubated with 2-deoxyglucose, which did not result in accumulation of glycogen. Immunocytochemical evidence indicates that glucose induces the aggregation of glycogen synthase molecules into clusters which are recovered in the sediments. These results indicate that glucose, in addition to activating glycogen synthase, may trigger changes in the localization of the enzyme in the cell.

Abstract: Disruption of the gene pgil of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level of glucose 6-phosphate was much higher in mutant than in wild-type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild-type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early-medium exponential cultures in wild-type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild-type cells at later stages. The fact that the intracellular levels of UDP-glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.

Abstract: Incubation of rat hepatocytes with glucose induces the translocation of glycogen synthase from soluble fractions to fractions which sediment at 10,000 g. Incubation of the cells with fructose, galactose, 2-deoxyglucose or 5-thioglucose, which activate glycogen synthase, also resulted in the translocation of the enzyme, whereas 3-O-methylglucose, 6-deoxyglucose and 1,5-anhydroglucitol, which do not cause the activation of the enzyme, were ineffective. Adenosine and carbonyl cyanide m-chlorophenylhydrazone, although activating glycogen synthase, did not induce its translocation. Mannoheptulose, which inhibits glucose phosphorylation, impaired the translocation of glycogen synthase induced by glucose. Furthermore, the extent of the translocation of the enzyme triggered by glucose and other sugars showed a high positive correlation with the intracellular concentration of glucose 6-phosphate. Microcystin, which blocks the activation of glycogen synthase by glucose, but not the accumulation of glucose 6-phosphate, did not affect the translocation of the enzyme. These results indicate that glucose 6-phosphate plays a role in the translocation of glycogen synthase in rat hepatocytes.

Abstract: The gene SIT4 of S. cerevisiae, which codes for a protein structurally related to the catalytic subunit of mammalian protein phosphatase 2A, was disrupted in vitro. Analysis of glycogen synthase activity ratio in mutant haploid cells indicated that the enzyme was less active than in wild-type cells. On the contrary, glycogen phosphorylase alpha activity was much higher. The activation of glycogen synthase observed in wild-type cells after incubation with lithium ions was not detected in mutant cells. These results suggest that the product of gene SIT4, a putative protein phosphatase, could be involved in the control of glycogen metabolism in yeast cells.

Abstract: S. cerevisiae gene DIS2S1, which codes for a protein very similar to the catalytic subunit of mammalian protein phosphatase 1, was disrupted "in vitro". Diploid yeast cells were transformed and sporulated. Tetrad analysis demonstrated that disruption of DIS2S1 is lethal for the cell. Glycogen phosphorylase alpha and glycogen synthase activity ratio were measured in diploids carrying a disrupted allele of the gene. Phosphorylase was dramatically activated in mutant cells but, under the same conditions, glycogen synthase activity was essentially identical in both mutant and wild-type cells.

Abstract: The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between 2.5 and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.

Abstract: Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.

Abstract: Although glycogen synthase is present in a highly inactivated state in hepatocytes from streptozocin-induced diabetic rats, glucagon, vasopressin, and vanadate are still able to further decrease the basal activity of the enzyme. This inactivation was observed with the low-to-high glucose 6-phosphate activity ratio assay. The inactivation of glycogen synthase occurred concomitantly with the activation of glycogen phosphorylase. When hepatocytes from diabetic rats were incubated with [32P]phosphate and then with the agents and when the 32P-labeled glycogen synthase was immunoprecipitated, we observed that the 32P bound to the 88,000-Mr subunit increased in all cases. All the [32P]phosphate was located in two cyanogen bromide fragments of the enzyme, indicating that the enzyme was phosphorylated at multiple sites. The fragments were precisely those phosphorylated by glycogenolytic hormones in hepatocytes from normal rats. These results demonstrated that hepatic glycogen synthase, although highly inactive, is under potential hormonal control in diabetes and that the enzyme has not reached its maximal level of phosphorylation. Furthermore, they indicated that vanadate behaves as a glycogenolytic agent regarding its effects on glycogen-metabolizing enzymes in hepatocytes from diabetic rats.

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Abstract: Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.

Abstract: Vanadate inactivated rat hepatocyte glycogen synthase and activated glycogen phosphorylase in a dose- and time-dependent manner. These effects were observed in hepatocytes from both fasted as well as fed rats. When rat hepatocytes were preincubated with [32P]phosphate and then with vanadate, and the 32P-labeled glycogen synthase was specifically immunoprecipitated, it was observed that vanadate stimulated the phosphorylation of the 88,000-dalton subunit of glycogen synthase. All of the phosphate was located in the same two CNBr fragments of the enzyme which are phosphorylated by glucagon and other glycogenolytic hormones. In cells incubated in a calcium-depleted medium, vanadate was still able to inactivate glycogen synthase but its effects on phosphorylase were essentially lost. These results demonstrate that, in the hepatocyte, vanadate exerts opposite effects than in the adipocyte and skeletal muscle, where vanadate has an insulin-like action.

Abstract: The Mr = 33,000 catalytic fragment of rabbit skeletal muscle type 1 protein phosphatase was digested with trypsin after reduction and alkylation. The resulting peptides were isolated, subjected to automated Edman degradation, and their sequences compared to the deduced peptide sequence of the bovine type 2A protein phosphatase cDNA. Of 10 tryptic peptides from the type 1 phosphatase that were sequenced, nine showed a high degree of homology with the type 2A phosphatase. This provides the first direct sequence comparison suggesting that the type 1 and type 2 protein phosphatases, distinguished functionally by their substrate specificities and sensitivity to inhibitors, make up part of a family of closely related gene products with similar structures.

Abstract: Incubation of rat hepatocytes with active phorbol esters and mezerein provoked a decrease in glycogen synthase activity. After the incubation of [3 2 P] phosphate-labeled cells with these tumor promoters, an increase in the amount of 3 2 P bound to the immunoprecipitated enzyme was observed. The decrease in activity highly correlated with the phosphorylation in the smaller CNBr fragment (CB-1) and only at high concentration of the phorbol ester the increase in the phosphorylation of the larger CNBr fragment (CB-2) became significative. Tryptic degradation of CB-1 showed two phosphopeptides after isoelectro focusing analysis (pI 3.9 and pI 3.4) and only one of them (pI 3.9) increased its phosphorylation state after treatment of the cells. These results indicate that the decrease in activity of glycogen synthase by phorbol esters and mezerein is a result of the phosphorylation of the enzyme and that a single site located in CB-1 is preferentially phosphorylated by these agents.

Abstract: Incubation of hepatocytes from fasted rats with LiCl provoked a concentration- and time-dependent activation of glycogen synthase. This effect was observed in the absence of glucose in the incubation medium. No changes in the intracellular concentrations of ATP or glucose-6-phosphate were detected. Lithium was also able to activate glycogen synthase in the absence of extracellular calcium. If hepatocytes were incubated with lithium and insulin, an additive effect of both agents on glycogen synthase activity was observed. LiCl was also effective in activating the enzyme in hepatocytes obtained from fed rats. When hepatocytes were incubated with [33P]phosphate and then treated with LiCl, a decrease in the amount of [32P]phosphate incorporated in the enzyme was observed. This dephosphorylation affected two CNBr fragments of the enzyme (CB-2 and CB-1), suggesting that several phosphorylation sites were involved. Lithium was also able to activate glycogen phosphorylase from both fasted and fed rats. Phosphorylase activation was concentration- and time-dependent, either in the presence or absence of calcium in the incubation medium. These findings demonstrate that although lithium appears to mimic the effects of insulin on glycogen synthase activity, its mechanism of action must be different from that of the hormone.

Abstract: 32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.

Abstract: The effects of epinephrine and vasopressin on the phosphorylation state of glycogen synthase were studied using rat hepatocytes incubated with 32P. After the incubation with hormones, 32P-labeled glycogen synthase was isolated using antibodies against rat liver enzyme. The immunoprecipitate showed a single radioactive band ( Mapp 88 kDa) when subjected to SDS-gel electrophoresis. Both epinephrine and vasopressin inactivated the enzyme and increased the 32P content of glycogen synthase. Cleavage of the immunoprecipitate with CNBr yielded two major 32P-labeled fragments of Mapp approximately 27 and 12 kDa. Both hormones increased the 32P content of both fragments. These results prove that epinephrine and vasopressin increase the phosphate content of the enzyme promoting its phosphorylation at multiple sites.