SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.     

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.     

Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult

We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h. Here we show that the endoplasmic reticulum (ER) chaperones BiP/Kar2p and Lhs1p and the mitochondrial chaperone Hsp78 were also upregulated at the physiological temperature during recovery from thermal insult. The heat shock element (HSE) in the KAR2 promoter was found to be sufficient to drive DUR. The unfolded protein element could also evoke DUR, albeit weakly, in the absence of a functional HSE. BiP/Kar2p functions in ER translocation and assists protein folding. Here we found that the synthesis of new BiP/Kar2p molecules was negligible for more than an hour after the shift of the cells from 50 degrees C to 24 degrees C. Concomitantly, ER translocation was blocked, suggesting that preexisting BiP/Kar2p molecules or other necessary proteins were not functioning. Translocation resumed concomitantly with enhanced synthesis of BiP/Kar2p after 3 h of recovery, after which ER exit and protein secretion also resumed. For a unicellular organism like S. cerevisiae, conformational repair of denatured proteins is the sole survival strategy. Chaperones that refold proteins in the cytosol, ER, and mitochondria of S. cerevisiae appear to be subject to DUR to ensure survival after thermal insults.     

Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris

One of the reasons for limited heterologous protein secretion in Pichia pastoris is the suboptimal folding conditions inside the cell. The Hsp70 and Hsp40 chaperone families in the cytoplasm or the ER regulate the folding and secretion of heterologous proteins. Here, we have studied the effect of chaperones Ydj1p, Ssa1p, Sec63p and Kar2p on the secretory expression of Candida antarctica lipase B (CalB) protein. Expression of CalB in P. pastoris resulted in the induction of Kar2p secretion into the medium surpassing the retrieval capacity of the cell. Individual overexpression of Ydj1p, Ssa1p and Sec63p in recombinant P. pastoris increased CalB expression level by 1.6-, 1.4- and 1.4-fold respectively compared to the control strain harboring only the CalB gene. However, overexpression of Kar2p had a negative effect on the expression of CalB. Moreover, Western blot analysis indicated accumulation and secretion of Kar2p in the ER, Golgi and extracellular medium in the chaperone coexpression strains. When expressed in combinations such as Ydj1p–Ssa1p, Ydj1p–Sec63p, Kar2p–Ssa1p, Kar2p–Sec63p, the expression level of CalB was increased by 2.5-, 1.5-, 1.5- and 1.5-fold respectively. Contrastingly, the Kar2p–Ydj1p combination resulted in decreased CalB secretion in the supernatant. From these results, we conclude that overexpression of Kar2p is not required for the secretion of CalB. Also, our work confirmed the synergistic effect of Ssa1p and Ydj1p chaperones in the expression of CalB.     

Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum.

KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family. Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion. Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation. In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p. First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations. Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1. Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels. Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E. coli HSP70, DnaK, and DnaJ. We propose that the interaction between these two proteins is critical to their function in protein translocation.     

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP

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.     

Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.     

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.     

Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.     

Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding

The 70-kDa heat shock protein (Hsp70) family of molecular chaperones cooperates with cofactors to promote protein folding, assembly of protein complexes and translocation of proteins across membranes. Although many cofactors of cytosolic Hsp70s have been identified, knowledge about cofactors of BiP/Kar2p, an endoplasmic reticulum (ER)-resident Hsp70, is still poor. Here we propose the Saccharomyces cerevisiae protein Rot1p as a possible cofactor of BiP/Kar2p involved in protein folding. Rot1p was found to be an essential, ER-localized membrane protein facing the lumen. ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response. Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant. These findings suggest that Rot1p participates in protein folding with Kar2p. Morphological analysis of rot1-2 cells revealed cell wall defects and accumulation of autophagic bodies in the vacuole. This implies that the protein folding machinery in which Rot1p is involved chaperones proteins acting in various physiological processes including cell wall synthesis and lysis of autophagic bodies.     

Role of the unfolded protein response pathway in secretory stress and regulation of INO1 expression in Saccharomyces cerevisiae

The unfolded protein response pathway (UPR) enables the cell to cope with the buildup of unfolded proteins in the endoplasmic reticulum (ER). UPR loss-of-function mutants, hac1Delta and ire1Delta, are also inositol auxotrophs, a phenotype associated with defects in expression of INO1, the most highly regulated of a set of genes encoding enzymes of phospholipid metabolism. We now demonstrate that the UPR plays a functional role in membrane trafficking under conditions of secretory stress in yeast. Mutations conferring a wide range of membrane trafficking defects exhibited negative genetic interaction when combined with ire1Delta and hac1Delta. At semipermissive temperatures, carboxypeptidase Y transit time to the vacuole was slower in Sec(-) cells containing an ire1Delta or hac1Delta mutation than in Sec(-) cells with an intact UPR. The UPR was induced in Sec(-) cells defective in subcellular membrane trafficking events ranging from ER vesicle trafficking to distal secretion and in erg6Delta cells challenged with brefeldin A. However, the high levels of UPR induction observed under these conditions were not correlated with elevated INO1 expression. Indeed, many of the Sec(-) mutants that had elevated UPR expression at semipermissive growth temperatures failed to achieve wild-type levels of INO1 expression under these same conditions.