The diet-tissue discrimination factor is the amount by which a consumer’s tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.
Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, and biochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinary functional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones to form a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all three kingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteins diverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversification and thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimental analyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, which emerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modeling revealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However, despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in the translocation of proteins from the cytosol into mitochondria. Apj1’s specialized role is related to degradation of sumolyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specialized functions, allowing expansion of the chaperone functional network, while maintaining the overall structural organization of their parental gene.
J-protein; Hsp70; gene duplication; yeast; Hsp40; divergent evolution
The C-terminal 69 residues of the J-protein Zuo1 are sufficient to activate Pdr1, a transcription factor involved in both pleiotropic drug resistance (PDR) and growth control. Little is understood about the pathway of activation by this primarily ribosome-associated Hsp40 co-chaperone. Here we report that only the C-terminal 13 residues of Zuo1 are required for activation of Pdr1, with hydrophobic residues being critical for activity. Two-hybrid interaction experiments suggest that the interaction between this 13-residue Zuo1 peptide and Pdr1 is direct, analogous to the activation of Pdr1 by xenobiotics. However, simply dissociation of Zuo1 from the ribosome is not sufficient for induction of Pdr1 transcriptional activity, as the C-terminal 86 residues of Zuo1 fold into an autoinhibitory left-handed four-helix bundle. Hydrophobic residues critical for interaction with Pdr1 are sequestered within the structure of this C-terminal domain (CTD), necessitating unfolding for activation. Thus, although expression of the CTD does not result in activation, alterations that destabilize the structure cause induction of PDR. These destabilizing alterations also result in dissociation of the full-length protein from the ribosome. Thus, our results are consistent with an activation pathway in which unfolding of Zuo1’s C-terminal helical bundle domain results in ribosome dissociation followed by activation of Pdr1 via a direct interaction.
molecular chaperone; Hsp40; PDR; pleiotropic drug resistance; ribosome
Hsp70 molecular chaperones are ubiquitous. By preventing aggregation, promoting folding, and regulating degradation, Hsp70s are major factors in the ability of cells to maintain proteostasis. Despite a wealth of functional information, little is understood about the evolutionary dynamics of Hsp70s. We undertook an analysis of Hsp70s in the fungal clade Ascomycota. Using the well-characterized 14 Hsp70s of Saccharomyces cerevisiae, we identified 491 orthologs from 53 genomes. Saccharomyces cerevisiae Hsp70s fall into seven subfamilies: four canonical-type Hsp70 chaperones (SSA, SSB, KAR, and SSC) and three atypical Hsp70s (SSE, SSZ, and LHS) that play regulatory roles, modulating the activity of canonical Hsp70 partners. Each of the 53 surveyed genomes harbored at least one member of each subfamily, and thus establishing these seven Hsp70s as units of function and evolution. Genomes of some species contained only one member of each subfamily that is only seven Hsp70s. Overall, members of each subfamily formed a monophyletic group, suggesting that each diversified from their corresponding ancestral gene present in the common ancestor of all surveyed species. However, the pattern of evolution varied across subfamilies. At one extreme, members of the SSB subfamily evolved under concerted evolution. At the other extreme, SSA and SSC subfamilies exhibited a high degree of copy number dynamics, consistent with a birth–death mode of evolution. KAR, SSE, SSZ, and LHS subfamilies evolved in a simple divergent mode with little copy number dynamics. Together, our data revealed that the evolutionary history of this highly conserved and ubiquitous protein family was surprising complex and dynamic.
molecular chaperones; multi gene family; gene duplication; birth-and-death; concerted evolution; Ascomycota genomes
Faithful replication and propagation of mitochondrial DNA (mtDNA) is critical for cellular respiration. Molecular chaperones, ubiquitous proteins involved in protein folding and remodeling of protein complexes, have been implicated in mtDNA transactions. In particular, cells lacking Mdj1, an Hsp40 co-chaperone of Hsp70 in the mitochondrial matrix, do not maintain functional mtDNA. Here we report that the great majority of Mdj1 is associated with nucleoids, DNA-protein complexes that are the functional unit of mtDNA transactions. Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA. However, a J-domain containing fragment expressed at the level that Mdj1 is normally present is not competent to maintain mtDNA, suggesting a function of Mdj1 beyond that carried out by its J-domain. Nevertheless, loss of mtDNA function upon Mdj1 depletion is retarded when the J-domain, is overexpressed. Analysis of Mdj1 variants revealed a correlation between nucleoid association and DNA maintenance activity, suggesting that localization is functionally important. We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association. Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.
•J-protein co-chaperone Mdj1 is critical for maintenance of functional mtDNA.•Majority of Mdj1 localizes to the nucleoid, likely via interaction with mtDNA.•Nucleoid localization of Mdj1 is necessary for mtDNA maintenance.•Function of Mdj1 in mtDNA maintenance requires cooperation with Hsp70.
J-protein; Molecular chaperone; DNA transactions; Yeast
The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis.
Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ+], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI+] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.
Hsp40; Ssa; heat-shock; protein misfolding; cell stress; Hsp104; PIN; saccharomyces; neurodegenerative disease
Mitochondria are essential organelles required for a number of key cellular processes. As most mitochondrial proteins are nuclear encoded, their efficient translocation into the organelle is critical. Transport of proteins across the inner membrane is driven by a multicomponent, matrix-localized “import motor,” which is based on the activity of the molecular chaperone Hsp70 and a J-protein cochaperone. In Saccharomyces cerevisiae, two paralogous J-proteins, Pam18 and Mdj2, can form the import motor. Both contain transmembrane and matrix domains, with Pam18 having an additional intermembrane space (IMS) domain. Evolutionary analyses revealed that the origin of the IMS domain of S. cerevisiae Pam18 coincides with a gene duplication event that generated the PAM18/MDJ2 gene pair. The duplication event and origin of the Pam18 IMS domain occurred at the relatively ancient divergence of the fungal subphylum Saccharomycotina. The timing of the duplication event also corresponds with a number of additional functional changes related to mitochondrial function and respiration. Physiological and genetic studies revealed that the IMS domain of Pam18 is required for efficient growth under anaerobic conditions, even though it is dispensable when oxygen is present. Thus, the gene duplication was beneficial for growth capacity under particular environmental conditions as well as diversification of the import motor components.
J-protein; mitochondria; protein translocation; Hsp70; gene duplication
Pam18, the J-protein cochaperone of the Hsp70-based mitochondrial import motor, forms a heterodimer with the structurally related protein Pam16. Genetic and biochemical studies suggest a critical role of this interaction in maintaining Pam18's association with the translocon rather than its previously proposed regulatory role.
The heat-shock protein 70 (Hsp70)–based import motor, associated with the translocon on the matrix side of the mitochondrial inner membrane, drives translocation of proteins via cycles of binding and release. Stimulation of Hsp70's ATPase activity by the translocon-associated J-protein Pam18 is critical for this process. Pam18 forms a heterodimer with the structurally related protein Pam16, via their J-type domains. This interaction has been proposed to perform a critical regulatory function, inhibiting the ATPase stimulatory activity of Pam18. Using biochemical and genetic assays, we tested this hypothesis by assessing the in vivo function of Pam18 variants having altered abilities to stimulate Hsp70's ATPase activity. The observed pattern of genetic interactions was opposite from that predicted if the heterodimer serves an inhibitory function; instead the pattern was consistent with that of mutations known to cause reduction in the stability of the heterodimer. Analysis of a previously uncharacterized region of Pam16 revealed its requirement for formation of an active Pam18:Pam16 complex able to stimulate Hsp70's ATPase activity. Together, our data are consistent with the idea that Pam18 and Pam16 form a stable heterodimer and that the critical role of the Pam18:Pam16 interaction is the physical tethering of Pam18 to the translocon via its interaction with Pam16.
Hsp70s, ubiquitous molecular chaperones, function in a myriad of biological processes, modulating polypeptides’ folding, degradation and translocation across membranes, as well as protein-protein interactions. This multitude of roles is not easily reconciled with the near conformity of biochemical activity of Hsp70s, an ATP-dependent client protein binding/release cycle. Much of the functional diversity of Hsp70s is driven by a diverse class of cofactors, J-proteins (also called Hsp40s). Often, multiple J-proteins function with a single Hsp70. Some target Hsp70 activity to clients at precise locations in cells; others bind client proteins directly, thereby delivering specific clients to Hsp70, directly determining their fate.
Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these “nouveau prions” has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI+], differs from the best studied, archetypal prion [PSI+] in several significant ways.1 Notably, [SWI+] is highly sensitive to alterations in Hsp70 system chaperone activity and is lost upon growth at elevated temperatures. In that report we briefly noted a correlation amongst prions regarding amino acid composition, seed number and sensitivity to the activity of the Hsp70 chaperone system. Here we extend that analysis and put forth the idea that [SWI+] may be representative of a class of asparagine-rich yeast prions which also includes [URE3], [MOT3+] and [ISP+], distinct from the glutamine-rich prions such as [PSI+] and [RNQ+]. While much work remains, it is apparent that our understanding of the extent of the diversity of prion characteristics is in its infancy.
Sis1; Hsp40; chromatin remodeling; Swi1; Ssa; heat-shock; protein misfolding; cell stress; Hsp 104; PIN
Frataxin is a highly conserved mitochondrial protein whose deficiency in humans results in Friedreich’s ataxia (FRDA), an autosomal recessive disorder characterized by progressive ataxia and cardiomyopathy. Although its cellular function is still not fully clear, the fact that frataxin plays a crucial role in Fe-S assembly on the scaffold protein Isu is well accepted. Here we report the characterisation of eight frataxin variants having alterations on two putative functional regions – the α1/β1 acidic ridge and the conserved β-sheet surface. We report that frataxin iron binding capacity is quite robust: even when five of the most conserved residues from the putative iron binding region are altered, at least 2 iron atoms per monomer can be bound, although with decreased affinity. Furthermore, we conclude that the acidic ridge is designed to favour function over stability. The negative charges have a functional role, but at the same time significantly impair frataxin’s stability. Removing five of those charges results in a thermal stabilization of ~24°C and reduces the inherent conformational plasticity. Alterations on the conserved β-sheet residues have only a modest impact on the protein stability, highlighting the functional importance of residues 122-124.
yeast frataxin; protein folding; metallochaperone; binding affinities; protein plasticity
The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.
Yeast prions are heritable genetic elements, formed spontaneously by aggregation of a single protein. Prions can thus generate diverse phenotypes in a dominant, non-Mendelian fashion, without a corresponding change in chromosomal gene structure. Since the phenotypes caused by the presence of a prion are thought to affect the ability of cells to survive under different environmental conditions, those that have global effects on cell physiology are of particular interest. Here we report the results of a study of one such prion, [SWI+], formed by a component of the SWI/SNF chromatin-remodeling complex, which is required for the regulation of a diverse set of genes. We found that, compared to previously well-studied prions, [SWI+] is highly sensitive to changes in the activities of molecular chaperones, particularly components of the Hsp70 machinery. Both under- and over-expression of components of this system initiated rapid loss of the prion from the cell population. Since expression of molecular chaperones, often known as heat shock proteins, are known to vary under diverse environmental conditions, such “chaperone sensitivity” may allow alteration of traits that under particular environmental conditions convey a selective advantage and may be a common characteristic of prions formed from proteins involved in global gene regulation.
J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.
Cytosolic chaperones are a diverse group of ubiquitous proteins that play central roles in multiple processes within the cell, including protein translation, folding, intracellular trafficking, and quality control. These cellular proteins have also been implicated in the replication of numerous viruses, although the full extent of their involvement in viral replication is unknown. We have previously shown that the heat shock protein 40 (hsp40) chaperone encoded by the yeast YDJ1 gene facilitates RNA replication of flock house virus (FHV), a well-studied and versatile positive-sense RNA model virus. To further explore the roles of chaperones in FHV replication, we examined a panel of 30 yeast strains with single deletions of cytosolic proteins that have known or hypothesized chaperone activity. We found that the majority of cytosolic chaperone deletions had no impact on FHV RNA accumulation, with the notable exception of J-domain-containing hsp40 chaperones, where deletion of APJ1 reduced FHV RNA accumulation by 60%, while deletion of ZUO1, JJJ1, or JJJ2 markedly increased FHV RNA accumulation, by 4- to 40-fold. Further studies using cross complementation and double-deletion strains revealed that the contrasting effects of J domain proteins were reproduced by altering expression of the major cytosolic hsp70s encoded by the SSA and SSB families and were mediated in part by divergent effects on FHV RNA polymerase synthesis. These results identify hsp70 chaperones as critical regulators of FHV RNA replication and indicate that cellular chaperones can have both positive and negative regulatory effects on virus replication.
Isu, the scaffold protein on which Fe-S clusters are built in the mitochondrial matrix, plays a central role in the biogenesis of Fe-S cluster proteins. We report that the reduction in the activity of several components of the cluster biogenesis system, including the specialized Hsp70 Ssq1, causes a 15–20-fold up-regulation of Isu. This up-regulation results from changes at both the transcriptional and posttranslational level: an increase in ISU mRNA levels and in stability of ISU protein. Its biological importance is demonstrated by the fact that cells lacking Ssq1 grow poorly when Isu levels are prevented from rising above those found in wild-type cells. Of the biogenesis factors tested, Nfs1, the sulfur donor, was unique. Little increase in Isu levels occurred when Nfs1 was depleted. However, its presence was required for the up-regulation caused by reduction in activity of other components. Our results are consistent with the existence of a mechanism to increase the stability of Isu, and thus its level, that is dependent on the presence of the cysteine desulfurase Nfs1.
Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.
Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18IMS) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon.
The Hsp70 Ssb and J protein Zuo1 of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, proposed to be involved in the folding of newly synthesized polypeptide chains. Cells lacking Ssb and/or Zuo1 have been reported to be hypersensitive to cationic aminoglycoside protein synthesis inhibitors that affect translational fidelity and to NaCl. Since we found that Δssb1 Δssb2 (Δssb1,2), Δzuo1, and wild-type cells have very similar levels of translational misreading in the absence of aminoglycosides, we asked whether the sensitivities to aminoglycosides and NaCl represent a general increase in sensitivity to cations. We found that Δssb1,2 and Δzuo1 cells are hypersensitive to a wide range of cations. This broad sensitivity is similar to that of cells having lowered activity of major plasma membrane transporters, such as the major K+ transporters Trk1 and Trk2 or their regulators Hal4 and Hal5. Like Δhal4,5 cells, Δssb1,2 and Δzuo1 cells have increased intracellular levels of Na+ and Li+ upon challenge with higher-than-normal levels of these cations, due to an increased rate of influx. In the presence of aminoglycosides, Δssb1,2, Δzuo1, and Δhal 4,5 cells have similarly increased levels of translational misreading. We conclude that, in vivo, the major cause of the aminoglycoside sensitivity of cells lacking ribosome-associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins.
Sis1 and Ydj1, functionally distinct heat shock protein (Hsp)40 molecular chaperones of the yeast cytosol, are homologs of Hdj1 and Hdj2 of mammalian cells, respectively. Sis1 is necessary for propagation of the Saccharomyces cerevisiae prion [RNQ+]; Ydj1 is not. The ability to function in [RNQ+] maintenance has been conserved, because Hdj1 can function to maintain Rnq1 in an aggregated form in place of Sis1, but Hdj2 cannot. An extended glycine-rich region of Sis1, composed of a region rich in phenylalanine residues (G/F) and another rich in methionine residues (G/M), is critical for prion maintenance. Single amino acid alterations in a short stretch of amino acids of the G/F region of Sis1 that are absent in the otherwise highly conserved G/F region of Ydj1 cause defects in prion maintenance. However, there is some functional redundancy within the glycine-rich regions of Sis1, because a deletion of the adjacent glycine/methionine (G/M) region was somewhat defective in propagation of [RNQ+] as well. These results are consistent with a model in which the glycine-rich regions of Hsp40s contain specific determinants of function manifested through interaction with Hsp70s.
G proteins, which bind and hydrolyze GTP, are involved in regulating a variety of critical cellular processes, including the process of protein synthesis. Many members of the subfamily of elongation factor class G proteins interact with the ribosome and function to regulate discrete steps during the process of protein synthesis. Despite sequence similarity to factors involved in translation, a role for the yeast Hbs1 protein has not been defined. In this work we have identified a genetic relationship between genes encoding components of the translational apparatus and HBS1. HBS1, while not essential for viability, is important for efficient growth and protein synthesis under conditions of limiting translation initiation. The identification of an Hbs1p-interacting factor, Dom34p, which shares a similar genetic relationship with components of the translational apparatus, suggests that Hbs1p and Dom34p may function as part of a complex that facilitates gene expression. Dom34p contains an RNA binding motif present in several ribosomal proteins and factors that regulate translation of specific mRNAs. Thus, Hbs1p and Dom34p may function together to help directly or indirectly facilitate the expression either of specific mRNAs or under certain cellular conditions.
In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.
In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding. However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained. As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes. Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions. Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain. We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.
Ydj1; Sis1; molecular chaperone; heat shock protein 70; yeast
The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Δssq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389–18393, 1998). However, the intermediate form in both wild-type and Δssq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Δssq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Δssq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Δssq1 mitochondria. Δssq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.