Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.     

Molecular chaperones and the assembly of the prion Sup35p, an in vitro study

The protein Sup35 from Saccharomyces cerevisiae possesses prion properties. In vivo, a high molecular weight form of Sup35p is associated to the [PSI+] factor. The continued propagation of [PSI+] is highly dependent on the expression levels of molecular chaperones from the Hsp100, 70 and 40 families; however, so far, their role in this process is unclear. We have developed a reproducible in vitro system to study the effects of molecular chaperones on the assembly of full-length Sup35p. We show that Hsp104p greatly stimulates the assembly of Sup35p into fibrils, whereas Ydj1p has inhibitory effect. Hsp82p, Ssa1p and Sis1p, individually, do not affect assembly. In contrast, Ssa1p together with either of its Hsp40 cochaperones blocks Sup35p polymerization. Furthermore, Ssa1p and Ydj1p or Sis1p can counteract the stimulatory activity of Hsp104p, by forming complexes with Sup35p oligomers, in an ATP-dependent manner. Our observations reveal the functional differences between Hsp104p and the Hsp70-40 systems in the assembly of Sup35p into fibrils and bring new insight into the mechanism by which molecular chaperones influence the propagation of [PSI+].     

Molecular chaperones and the assembly of the prion Ure2p in vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

分子伴侣对可溶性肿瘤坏死因子相关促凋亡配体在巴斯德毕赤酵母中表达的影响

目的:提高外源蛋白可溶性肿瘤坏死因子相关促凋亡配体(sTRAIL)在巴斯德毕赤酵母中的分泌表达。方法:根据GenBank公共数据库中公布的模式生物酿酒酵母的分子伴侣(Ssa1p、YDJ1、Kar2p和PDI)基因序列设计引物,利用PCR方法从酿酒酵母基因组中得到各基因片段,并将单独Ssa1p或Kar2p、组合YDJ1 PDI、Kar2p PDI或YDJ1 PDI PDI分别构建到pPIB2Z表达载体中,并整合到外源蛋白sTRAIL工程菌(毕赤酵母GS115)中进行筛选和诱导表达。结果:SDS-PAGE分析表明,sTRAIL的表达量明显提高,特别是整合了分子伴侣组合YDJ1 PDI的工程菌。Western印迹分析整合的分子伴侣基因后,分子伴侣蛋白在工程菌中的表达量得到了提高。结论:提高细胞内分子伴侣的表达,可以增加外源蛋白的分泌表达,为进一步研究巴斯德毕赤酵母奠定了基础。     

Sti1 is a novel activator of the Ssa proteins

The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.     

Disassembling protein aggregates in the yeast cytosol. The cooperation of Hsp26 with Ssa1 and Hsp104

In all organisms studied, elevated temperatures induce the expression of a variety of stress proteins, among them small Hsps (sHsp). sHsps are chaperones that prevent the unspecific aggregation of proteins by forming stable complexes with unfolded polypeptides. Reactivation of captured proteins requires the assistance of other ATP-dependent chaperones. How sHsps and ATP-dependent chaperones work together is poorly understood. Here, we analyzed the interplay of chaperones present in the cytosol of Saccharomyces cerevisiae. Specifically, we characterized the influence of Hsp104 and Ssa1 on the disassembly of Hsp26 x substrate complexes in vitro and in vivo. We show that recovery of proteins from aggregates in the cell requires the chaperones to work together with defined but overlapping functions. During reactivation, proteins are transferred from a stable complex with Hsp26 to Hsp104 and Hsp70. The need for ATP-dependent chaperones depends on the type of sHsp x substrate complex. Although Ssa1 is able to release substrate proteins from soluble Hsp26 x substrate complexes, Hsp104 is essential to dissociate substrate proteins from aggregates with incorporated sHsps. Our results are consistent with a model of several interrelated defense lines against protein aggregation.     

Cns1 is an activator of the Ssa1 ATPase activity

Hsp90 is a key mediator in the folding process of a growing number of client proteins. The molecular chaperone cooperates with many co-chaperones and partner proteins to fulfill its task. In Saccharomyces cerevisiae, several co-chaperones of Hsp90 interact with Hsp90 via a tetratricopeptide repeat (TPR) domain. Here we show that one of these proteins, Cns1, binds both to Hsp90 and to the yeast Hsp70 protein Ssa1 with comparable affinities. This is reminiscent of Sti1, another TPR-containing co-chaperone. Unlike Sti1, Cns1 exhibits no influence on the ATPase of Hsp90. However, it activates the ATPase of Ssa1 up to 30-fold by accelerating the rate-limiting ATP hydrolysis step. This stimulating effect is mediated by the N-terminal TPR-containing part of Cns1, whereas the C-terminal part showed no effect. Competition experiments allow the conclusion that Hsp90 and Ssa1 compete for binding to the single TPR domain of Cns1. Taken together, Cns1 is a potent cochaperone of Ssa1. Our findings highlight the importance of the regulation of Hsp70 function in the context of the Hsp90 chaperone cycle.     

Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.     

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.     

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.     

Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co-chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non-native protein substrate. Here, we show that members of the Hsp70-related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae, Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome-associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N-terminal ATPase domain and the ultimate C-terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70-mediated re-folding of thermally denatured firefly luciferase in vitro. Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo. Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.     

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.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.     

Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae

We have found that the guanine nucleotide exchange factor for ras, Cdc25p, interacts with Ssa1p in Saccharomyces cerevisiae. This interaction was observed with GST-fused Cdc25p polypeptides and confirmed by coimmunoprecipitation with the endogenous Cdc25p. Hsp82 appeared also to be co-immunoprecipitated with Cdc25p, albeit to a lower level than Hsp70. In a strain deleted for SSA1 and SSA2, we observed a reduced cellular content of Cdc25p. Consistent with a reduced activity of the cAMP-dependent PKA pathway, the rate of accumulation of both trehalose and glycogen was stimulated in the ssa-deleted strain. Expression of SSA1 reversed these effects, whereas co-expression of SSA1 and PDE2 restored high accumulation. The expression of genes repressed by cAMP, GAC1 and TPS1, fused to beta-galactosidase, was also stimulated by deletion of SSA genes. The effect of ssa deletion on glycogen accumulation was lost in a strain deleted for CDC25 rescued by the RAS2ile152 allele. Altogether, these results lead to the conclusion that Ssa1p positively controls the cAMP pathway through Cdc25p. We propose that this connection plays a critical role in the adaptation of cells to stress conditions.     

Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro. Based on the strict positional conservation of the Arg-237 residue, several Bag domain proteins were predicted from various eukaryotic genomes. One candidate, Snl1p from Saccharomyces cerevisiae, was confirmed as a Bag domain co-chaperone. Snl1p bound specifically to the Ssa and Ssb forms of yeast cytosolic Hsp70, as revealed by two-hybrid screening and co-precipitations from yeast lysate. In vitro, Snl1p also recognized mammalian Hsp70 and regulated the Hsp70 ATPase activity identically to Bag-1M. Point mutations in Snl1p that disrupted the conserved residues Glu-112 and Arg-141, equivalent to Glu-212 and Arg-237 in Bag-1M, abolished the interaction with Hsp70 proteins. In live yeast, mutated Snl1p could not substitute for wild-type Snl1p in suppressing the lethal defect caused by truncation of the Nup116p nuclear pore component. Thus, Snl1p is the first Bag domain protein identified in S. cerevisiae, and its interaction with Hsp70 is essential for biological activity.     

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO-flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo1p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p.