Abstract: The carboxyl terminus of Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone and a U-box ubiquitin ligase that plays a crucial role in protein quality control in higher eukaryotes. The yeast Yarrowia lipolytica is the only known hemiascomycete where a CHIP ortholog is found. Here, we characterize Y. lipolytica's CHIP ortholog (Yl.Chn1p) and document its interactions with components of the protein quality control machinery. We show that Yl.Chn1p is non-essential unless Y. lipolytica is severely stressed. We sought for genetic interactions among key components of the Y. lipolytica protein quality control arsenal, including members of the Ssa-family of Hsp70 molecular chaperones, the Yl.Bag1p Hsp70 nucleotide exchange factor, the Yl.Chn1p and Yl.Ufd2p U-box ubiquitin ligases, the Yl.Doa10p and Yl.Hrd1p RING-finger ubiquitin ligases, and the Yl.Hsp104p disaggregating molecular chaperone. Remarkably, no synthetic phenotypes were observed among null alleles of the corresponding genes in most cases, suggesting that overlapping pathways efficiently act to enable Y. lipolytica cells to survive under harsh conditions. Yl.Chn1p interacts with mammalian and Saccharomyces cerevisiae members of the Hsp70 family in vitro, and these interactions are differently regulated by Hsp70 co-chaperones. We demonstrate notably that Yl.Chn1p/Ssa1p interaction is Fes1p-dependent and the formation of an Yl.Chn1p/Ssa1p/Sse1p ternary complex. Finally, we show that, similar to Sse1p, Yl.Chn1p can act as a "holdase" to prevent the aggregation of a heat-denatured protein.
Abstract: The epigenetic factor [PSI(+) ] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI(+) ] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI(+) ] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI(+) ] and weakening of [PSI(+) ] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI(+) ] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.
Abstract: The aggregation of α-synuclein (α-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native α-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with α-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and α-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble α-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble α-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing α-Syn assembly inhibition by Hsc70. We show that Hsc70 binds α-Syn fibrils with a 5-fold tighter affinity compared with soluble α-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight α-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of α-Syn assemblies. We show a reduced cellular toxicity of α-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of α-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.
Abstract: Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI (+) ], [URE3] and [PIN (+) ] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the "non-prion" domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.
Abstract: The yeast Saccharomyces cerevisiae is able to form complex multicellular structures called mats on low-density agar Petri plates. Mat formation strictly depends on Flo11p, a cell surface mannoprotein that mediates the adhesion of yeast cells to the agar surface. Here, we show that Swa2p, an auxilin ortholog required for clathrin-coated vesicle uncoating, is strictly required for biofilm formation. We show that the maturation and cellular levels of Flo11p are affected in Deltaswa2 cells, yet without compromising invasive growth. Both the TPR and J-domains of Swa2p, but not its clathrin-binding and ubiquitin-association motifs, are required for its function in Flo11p processing.
Abstract: Since their recent identification, eukaryotic Hsp70 nucleotide exchange factors (NEFs) have gained increasing interest due to their engagement in vital cellular processes. Here, I summarize our current knowledge of their mechanisms of action, regulations and cellular functions as well as their relevance for human diseases such as cystic fibrosis or amyloidoses.
Abstract: BACKGROUND: The cytosol of most eukaryotic cells contains multiple highly conserved Hsp70 orthologs that differ mainly by their spatio-temporal expression patterns. Hsp70s play essential roles in protein folding, transport or degradation, and are major players of cellular quality control processes. However, while several reports suggest that specialized functions of Hsp70 orthologs were selected through evolution, few studies addressed systematically this issue. METHODOLOGY/PRINCIPAL FINDINGS: We compared the ability of Ssa1p-Ssa4p from Saccharomyces cerevisiae and Ssa5p-Ssa8p from the evolutionary distant yeast Yarrowia lipolytica to perform Hsp70-dependent tasks when expressed as the sole Hsp70 for S. cerevisiae in vivo. We show that Hsp70 isoforms (i) supported yeast viability yet with markedly different growth rates, (ii) influenced the propagation and stability of the [PSI(+)] and [URE3] prions, but iii) did not significantly affect the proteasomal degradation rate of CFTR. Additionally, we show that individual Hsp70 orthologs did not induce the formation of different prion strains, but rather influenced the aggregation properties of Sup35 in vivo. Finally, we show that [URE3] curing by the overexpression of Ydj1p is Hsp70-isoform dependent. CONCLUSION/SIGNIFICANCE: Despite very high homology and overlapping functions, the different Hsp70 orthologs have evolved to possess distinct activities that are required to cope with different types of substrates or stress situations. Yeast prions provide a very sensitive model to uncover this functional specialization and to explore the intricate network of chaperone/co-chaperone/substrates interactions.
Abstract: Hsp70 molecular chaperones play a variety of functions in every organism, cell type and organelle, and their activities have been implicated in a number of human pathologies, ranging from cancer to neurodegenerative diseases. The functions, regulations and structure of Hsp70s were intensively studied for about three decades, yet much still remains to be learned about these essential folding enzymes. Genome sequencing efforts revealed that most genomes contain multiple members of the Hsp70 family, some of which co-exist in the same cellular compartment. For example, the human cytosol and nucleus contain six highly homologous Hsp70 proteins while the yeast Saccharomyces cerevisiae contains four canonical Hsp70s and three fungal-specific ribosome-associated and specialized Hsp70s. The reasons and significance of the requirement for multiple Hsp70s is still a subject of debate. It has been postulated for a long time that these Hsp70 isoforms are functionally redundant and differ only by their spatio-temporal expression patterns. However, several studies in yeast and higher eukaryotic organisms challenged this widely accepted idea by demonstrating functional specificity among Hsp70 isoforms. Another element of complexity is brought about by specific cofactors, such as Hsp40s or nucleotide exchange factors that modulate the activity of Hsp70s and their binding to client proteins. Hence, a dynamic network of chaperone/co-chaperone interactions has evolved in each organism to efficiently take advantage of the multiple cellular roles Hsp70s can play. We summarize here our current knowledge of the functions and regulations of these molecular chaperones, and shed light on the known functional specificities among isoforms.
Abstract: Following endoplasmic reticulum (ER) stress, eukaryotic cells trigger a conserved signal transduction pathway called the unfolded protein response (UPR) that regulates the ER's capacity to perform protein folding according to cellular demand. In Saccharomyces cerevisiae, the UPR is initiated by Ire1, a type I transmembrane serine/threonine kinase/endoribonuclease, that senses unfolded protein levels within the ER in collaboration with the ER Hsp70-family member, BiP/Kar2. Here, we report on the characterization of the Yarrowia lipolytica Ire1 ortholog. Our results show that Sls1, a nucleotide exchange factor for BiP, has important functions in regulating ER stress and the interaction of BiP and Ire1. They suggest that Sls1 regulates this interaction, by stimulating the conversion of BiP from the ADP-bound to the ATP-bound state, which favors its interaction with Ire1. Moreover, we identified known and new partners for Ire1 using the Tandem Affinity Purification (TAP) approach.
Abstract: The yeast Saccharomyces cerevisiae has been used as a model for fungal biofilm formation due to its ability to adhere to plastic surfaces and to form mats on low-density agar petri plates. Mats are complex multicellular structures composed of a network of cables that form a central hub from which emanate multiple radial spokes. This reproducible and elaborate pattern is indicative of a highly regulated developmental program that depends on specific transcriptional programming, environmental cues, and possibly cell-cell communication systems. While biofilm formation and sliding motility were shown to be strictly dependent on the cell-surface adhesin Flo11p, little is known about the cellular machinery that controls mat formation. Here we show that Hsp70 molecular chaperones play key roles in this process with the assistance of the nucleotide exchange factors Fes1p and Sse1p and the Hsp40 family member Ydj1p. The disruption of these cofactors completely abolished mat formation. Furthermore, complex interactions among SSA genes were observed: mat formation depended mostly on SSA1 while minor defects were observed upon loss of SSA2; additional mutations in SSA3 or SSA4 further enhanced these phenotypes. Importantly, these mutations did not compromise invasive growth or Flo11p expression, suggesting that Flo11p-independent pathways are necessary to form mats.
Abstract: The Anc1 protein co-purifies with general transcription factors, chromatin remodeling complexes, and histone modification enzymes and is required for efficient transcription in yeast. We show here that Anc1 interacts with Tsm1, Tfg1, Sth1, Ino80, and Sas3 that are, respectively, the catalytic subunits of the general transcription factors TFIID and TFIIF, of the chromatin remodeling complexes RSC and INO80, and of the histone H3-acetyltransferase complex NuA3. We show that Anc1 is required for growth on galactose as the sole carbon source, and that it is recruited to the UAS of the GAL1 gene after induction.
Abstract: Regulation of ribosome biogenesis is central to the control of cell growth. In rapidly growing yeast cells, ribosomal protein (RP) genes account for approximately one-half of all polymerase II transcription-initiation events, yet these genes are markedly and coordinately downregulated in response to a number of environmental stress conditions, or during the transition from fermentation to respiration. Although several conserved signalling pathways (TOR, RAS/protein kinase A and protein kinase C) impinge upon RP gene transcription, little is known about how initiation at these genes is controlled. Rap1 (refs 6, 7) and more recently Fhl1 (ref. 8) were shown to bind upstream of many RP genes. Here we show that the essential protein Ifh1 binds to and activates many RP gene promoters under optimal growth conditions in Saccharomyces cerevisiae. Ifh1 is recruited to RP gene promoters through the forkhead-associated domain of Fhl1. Ifh1 binding decreases when RP genes are downregulated either by TOR inhibition or nutrient depletion, and is restored after release from starvation or upon regulated induction of IFH1 expression. These findings indicate a central role for Ifh1 and Fhl1 in RP gene regulation.
Abstract: ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.
Abstract: Hsp70 molecular chaperones play a variety of functions in every organism, cell type and compartment, and their activities have been implicated in a number of disease states. Hsp70 activity relies on ATP binding and hydrolysis by the N-terminal domain, which modulates peptide binding and release by the C-terminal domain. In Escherichia coli, the Hsp70 family member DnaK is regulated by DnaJ, which stimulates its ATPase activity, and by GrpE, a nucleotide exchange factor that promotes ADP release. In eukaryotic cells, Hsp70 regulation is far more complex and many families of positive and negative regulators have been characterized, such as Bag-1 that was first identified as an anti-apoptotic Bcl2-binding protein. Whereas eukaryotic DnaJ homologs have extensively been studied, GrpE homolog are found only in the mitochondria or chloroplasts. In fact, until the discovery of Bag-1 in mammalian cells, nucleotide exchange factors were presumed absent from the eukaryotic cytosol and organelles that comprise the secretory pathway, such as the endoplasmic reticulum (ER). However, members of a novel class of nucleotide exchange factors in the ER and in the cytoplasm have recently been identified that act on the ER lumen and cytoplasmic Hsp70 proteins, respectively. Although first uncovered in yeast and named Sls1p and Fes1p, we report here that the class of Hsp70 nucleotide exchange factors defined by Sls1p and Fes1p is conserved from yeast to humans.
Abstract: We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.
Abstract: The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.
Abstract: The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.
