Brodsky JL, Werner ED, Dubas ME, Goeckeler JL, Kruse KB and McCracken AAThe requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. J. Biol. Chem. 274: 3453-3460

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/1999; 274(6):3453-60. DOI: 10.1074/jbc.274.6.3453
Source: PubMed


Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in
the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and
defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p
translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1Δ and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-α factor is defective inkar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in thekar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating
that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also
required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-α factor and A1PiZ were degraded normally, indicating
further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

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    • "Because the sec63-1 mutant is leaky, and the cells are compromised for translocation into the ER even at the permissive temperature, it remains unclear whether the effect of the mutation indicated a direct involvement of Sec63p in ERAD, or whether the effect was indirect. In the gene encoding the ER-lumenal Hsp70 Kar2p specific mutations had been identified that cause ERAD defects [21]. In addition, two other ER-lumenal J-proteins, Jem1p and Scj1p, are required for ERAD [22]. "
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    ABSTRACT: How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.
    PLoS ONE 12/2013; 8(12):e82058. DOI:10.1371/journal.pone.0082058 · 3.23 Impact Factor
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    • "On the basis of in vitro studies, these proteins are incapable of folding substrates on their own and instead have evolved a novel Hsp70 binding interface, which they use to form highly stable Hsp70@BULLETHsp110 heterodimers to enhance nucleotide exchange (Shaner et al. 2005; Yam et al. 2005; Dragovic et al. 2006; Raviol et al. 2006; Polier et al. 2008; Schuermann et al. 2008). However, the peptide binding domains of the Hsp110 family, while altered with respect to Hsp70, retain the ability to bind and protect unfolded polypeptides as exemplified by the yeast Ssa1 protein (Brodsky et al. 1999). This raises the possibility that this group may act in a manner similar to the client-binding J proteins to enhance substrate delivery and transfer to Hsp70 for subsequent folding . "
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    ABSTRACT: A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.
    Genetics 12/2011; 190(4):1157-95. DOI:10.1534/genetics.111.128033 · 5.96 Impact Factor
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    • "Volume 23 February 15, 2012 UPR target gene regulation in stress tolerance | 633 tion comes from findings that the UPR facilitates stress tolerance by activating ERAD and vacuolar pathways (Spear and Ng, 2003). Kar2p is required for ERAD but its role in the ER-to-vacuole pathway is unknown (Brodsky et al., 1999; Plemper and Wolf, 1999). The interplay of these pathways under stress conditions is illustrated in Figure 4. "
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    ABSTRACT: The unfolded protein response (UPR) monitors and maintains protein homeostasis in the endoplasmic reticulum (ER). In budding yeast, the UPR is a transcriptional regulatory pathway that is quiescent under normal conditions. Under conditions of acute ER stress, activation of UPR targets is essential for cell viability. How individual target genes contribute to stress tolerance is unclear. Uncovering these roles is hampered because most targets also play important functions in the absence of stress. To differentiate stress-specific roles from everyday functions, a single target gene was uncoupled from UPR control by eliminating its UPR-specific regulatory element. Through this approach, the UPR remains intact, aside from its inability to induce the designated target. Applying the strategy to the major ER chaperone Kar2p/BiP revealed the physiological function of increasing its cellular concentration. Despite hundreds of target genes under UPR control, we show that activation of KAR2 is indispensable to alleviate some forms of ER stress. Specifically, activation is essential to dispose misfolded proteins that are otherwise toxic. Surprisingly, induced BiP/Kar2p molecules are dedicated to alleviating stress. The inability to induce KAR2 under stress had no effect on its known housekeeping functions.
    Molecular biology of the cell 12/2011; 23(4):630-41. DOI:10.1091/mbc.E11-04-0297 · 4.47 Impact Factor
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