Yeast nucleoporins involved in passive nuclear envelope permeability

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.     

Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70

Heat shock cognate protein 70 (Hsc70) serves nuclear transport of several proteins as a molecular chaperone. We have recently identified a novel variant of human Hsc70, heat shock cognate protein 54 (Hsc54), that lacks amino acid residues 464-616 in the protein binding and variable domains of Hsc70. In the present study, we examined nucleocytoplasmic localization of Hsc70 and Hsc54 by using green fluorescent protein (GFP) fusions. GFP-Hsc70 is localized in both the cytoplasm and the nucleus at 37 degrees C and accumulated into the nucleolus/nucleus after heat shock, whereas GFP-Hsc54 always remained exclusively in the cytoplasm under these conditions. Mutation studies indicated that 20 amino acid residues of nuclear localization-related signals, which are missing in Hsc54 but are retained in Hsc70, are required for proper nuclear localization of Hsc70. We further found that Hsc54 contains a functional leucine-rich nuclear export signal (NES, (394)LDVTPLSL(401)) which is differently situated from the previously proposed NES in Saccharomyces cerevisiae Ssb1p. The cytoplasmic localization of Hsc54 was impaired by a mutation in NES as well as by a nuclear export inhibitor, leptomycin B, suggesting that Hsc54 is actively exported from the nucleus to the cytoplasm through a CRM1-dependent mechanism. In contrast, the nucleocytoplasmic localization of Hsc70 was not affected by the same mutation of NES or leptomycin B. These results suggest that the nuclear localization-related signal could functionally mask NES leading to prolonged retention of Hsc70 in the nucleus. An additional mechanism for unmasking the NES may regulate nucleocytoplasmic trafficking of Hsc70.     

Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p

The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae. Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.     

In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation

The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.     

The carrier Msn5p/Kap142p promotes nuclear export of the hsp70 Ssa4p and relocates in response to stress

Cytoplasmic hsp70s like yeast Ssa4p shuttle between nucleus and cytoplasm under normal growth conditions but accumulate in nuclei upon stress. This nuclear accumulation is only transient, and Ssa4p relocates to the cytoplasm when cells recover. We show here that Ssa4p nuclear export is independent of Xpol/Crm1 and identify the importin-beta family member Msn5p/Kap142p as the exporter for Ssa4p. In growing cells and in vitro, Msn5p and Ssa4p generate genuine export complexes that require Ran/Gsp1p-GTP. Furthermore, nucleoporin Nup82p, which plays a role in Msn5p-mediated transport, is necessary for efficient export of Ssa4p. In living cells, stress not only regulates Ssa4p localization, but also controls the distribution of Msn5p. Msn5p is concentrated in nuclei of unstressed cells, but appears in the cytoplasm upon exposure to ethanol, heat, starvation or severe oxidative stress. In addition, growth on non-fermentable carbon sources relocates a portion of Msn5p to the cytoplasm and leads to a partial nuclear accumulation of Ssa4p. Taken together, growth and stress conditions that localize the transporter Msn5p to the cytoplasm also induce the nuclear accumulation of its cargo Ssa4p.     

Functionally redundant isoforms of a yeast Hsp70 chaperone subfamily have different antiprion effects

Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomyces cerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p and Ssa4p are 80% identical to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI(+)] and [URE3] prions. When expressed as the only Ssa, Ssa1p antagonized [URE3] and Ssa2p antagonized [PSI(+)]. Ssa3p and Ssa4p influenced [URE3] and [PSI(+)] somewhat differently but overall their effects paralleled those of Ssa1p and Ssa2p, respectively. Additionally, Ssa3p suppressed a prion-inhibitory effect of elevated temperature. Our previously described Ssa1-21p mutant weakens [PSI(+)] in SSA1-21 SSA2 cells and abolishes it in SSA1-21 ssa2Delta cells. To test if the same mutation affected other prions or altered Ssa2p similarly, we compared effects of a constructed Ssa2-21p mutant and Ssa1-21p on both prions. Surprisingly, [URE3] was unaffected in SSA1-21 SSA2 cells and could propagate in SSA1-21 ssa2Delta cells. Ssa2-21p impaired [URE3] considerably and weakened [PSI(+)] strongly but in a manner distinct from Ssa1-21p, highlighting functional differences between these nearly identical Hsp70's. Our data uncover exquisite functional differences among isoforms of a highly homologous cytosolic Hsp70 subfamily and point to a possibility that variations in Hsp70 function that might improve fitness under optimal conditions are also important during stress.     

Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei. To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene. With this assay we have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei when cells enter stationary phase.     

Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation.

To study the intracellular localization of MAPKAP kinase 2 (MK2), which carries a putative bipartite nuclear localization signal (NLS), we constructed a green fluorescent protein-MAPKAP kinase 2 fusion protein (GFP-MK2). In transfected cells, this protein is located predominantly in the nucleus; unexpectedly, upon stress, it rapidly translocates to the cytoplasm. This translocation can be blocked by the p38 MAP kinase inhibitor SB203580, indicating its regulation by phosphorylation. Molecular mimicry of MK2 phosphorylation at T317 in GFP-MK2 led to a mutant which is located almost exclusively in the cytoplasm of the cell, whereas the mutant T317A shows no stress-induced redistribution. Since leptomycin B, which inhibits the interaction of exportin 1 with the Rev-type leucine-rich nuclear export signal (NES), blocks stress-dependent translocation of GFP-MK2, it is supposed that phosphorylation-induced export of the protein causes the translocation. We have identified the region responsible for nuclear export in MK2 which is partially overlapping with and C-terminal to the autoinhibitory motif. This region contains a cluster of hydrophobic amino acids in the characteristic spacing of a leucine-rich Rev-type NES which is necessary to direct GFP-MK2 to the cytoplasm. However, unlike the Rev-type NES, this region alone is not sufficient for nuclear export. The data obtained indicate that MK2 contains a constitutively active NLS and a stress-regulated signal for nuclear export. Keywords: nuclear export/nuclear import/protein phosphorylation/signal transduction/stress response     

Proline-rich Akt substrate of 40-kDa contains a nuclear export signal

The proline-rich Akt substrate of 40-kDa (PRAS40) has been linked to the regulation of the activity of the mammalian target of rapamycin complex 1 as well as insulin action. Despite these cytosolic functions, PRAS40 was originally identified as nuclear phosphoprotein in Hela cells. This study aimed to detail mechanisms and consequences of the nucleocytosolic trafficking of PRAS40. Sequence analysis identified a potential leucine-rich nuclear export signal (NES) within PRAS40. Incubation of A14 fibroblasts overexpressing human PRAS40 (hPRAS40) resulted in nuclear accumulation of the protein. Furthermore, mutation of the NES mimicked the effects of leptomycin B, a specific inhibitor of nuclear export, on the subcellular localization of hPRAS40. Finally, A14 cells expressing the NES-mutant showed impaired activation of components of the Akt-pathway as well as of the mTORC1 substrate p70 S6 kinase after insulin stimulation. This impaired insulin signaling could be ascribed to reduced protein levels of insulin receptor substrate 1 in cells expressing mutant NES. In conclusion, PRAS40 contains a functional nuclear export signal. Furthermore, enforced nuclear accumulation of PRAS40 impairs insulin action, thereby substantiating the function of this protein in the regulation of insulin sensitivity.     

A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking

Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm.     

The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb

There is growing evidence that members of the extended Hsp70 family of molecular chaperones, including the Hsp110 and Grp170 subgroups, collaborate in vivo to carry out essential cellular processes. However, relatively little is known regarding the interactions and cellular functions of Sse1, the yeast Hsp110 homolog. Through co-immunoprecipitation analysis, we found that Sse1 forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. Binding of Ssa or Ssb to Sse1 was mutually exclusive. The ATPase domain of Sse1 was found to be critical for interaction as inactivating point mutations severely reduced interaction with Ssa and Ssb. Sse1 stimulated Ssa1 ATPase activity synergistically with the co-chaperone Ydj1, and stimulation required complex formation. Ssa1 is required for post-translational translocation of the yeast mating pheromone alpha-factor into the endoplasmic reticulum. Like ssa mutants, we demonstrate that sse1delta cells accumulate prepro-alpha-factor, but not the co-translationally imported protein Kar2, indicating that interaction between Sse1 and Ssa is functionally significant in vivo. These data suggest that the Hsp110 chaperone operates in concert with Hsp70 in yeast and that this collaboration is required for cellular Hsp70 functions.     

FAK nuclear export signal sequences

Ubiquitously expressed focal adhesion kinase (FAK), a critical component in transducing signals from sites of cell contacts with extracellular matrix, was named after its typical localization in focal adhesions. A nuclear localization of FAK has been also reported and its scaffolding role in nucleus and requirement for p53 ubiquitination were only recently described. Whereas FAK nuclear localization signal (NLS) was found in F2 lobe of FERM domain, nuclear export signal (NES) sequences have not been yet determined. Here we demonstrate that FAK has two NES sequences, NES1 in F1 lobe of FERM domain and NES2 in kinase domain. Although, both NES1 and NES2 are evolutionary conserved, and present as well in FAK-related protein kinase Pyk2, only NES2 demonstrates full biological nuclear export activity.     

Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.     

Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.     

Characterization of the nuclear export signal of polypyrimidine tract-binding protein.

The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, we have shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for its activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.     

Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.     

Feedback control of prion formation and propagation by the ribosome‐associated chaperone complex

Cross‐beta fibrous protein aggregates (amyloids and amyloid‐based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI⁺], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome‐associated complex (RAC), composed of Hsp40‐Zuo1 and Hsp70‐Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC‐deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed because of lack of ribosome‐associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild‐type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb‐based regulatory circuit.