No Country for Old Misfolded Glycoproteins
2011; Elsevier BV; Volume: 42; Issue: 6 Linguagem: Inglês
10.1016/j.molcel.2011.06.004
ISSN1097-4164
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoGauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar present evidence that an endoplasmic reticulum (ER) sugar-removing enzyme with a folding sensor subunit enables a stochastic quality control mechanism, which marks with increasing probability misfolded glycoproteins for destruction the longer they reside in the ER. Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar present evidence that an endoplasmic reticulum (ER) sugar-removing enzyme with a folding sensor subunit enables a stochastic quality control mechanism, which marks with increasing probability misfolded glycoproteins for destruction the longer they reside in the ER. On several occasions in Cormac McCarthy's novel No Country for Old Men, the character of Anton Chigurh (pronounced “sugar”) balances his murderous code against the capricious outcome of a coin toss. Heads, the potential victim lives; tails, he or she gets the cattle gun. With apologies, certain quality control mechanisms in the cell also have to make life/death decisions when deciding the fate of proteins attempting to fold (Wickner et al., 1999Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (891) Google Scholar). This is because the intracellular milieu is crowded, and folding intermediates that persistently expose their hydrophobic regions, normally buried in their native structures, will inevitably cause protein aggregation. Consequently, failure to efficiently target terminally misfolded proteins for destruction leads to many human disorders associated with the formation of protein aggregates. The opposite extreme is no good either: overzealous destruction of newly synthesized proteins that were on their way to becoming folded would potentially reduce cellular fitness due to the high energetic cost of protein biosynthesis. Thus, unlike Anton Chigurh, quality control mechanisms don't appear to make nihilistic decisions when they encounter misfolded proteins. Rather, they somehow “know” which of the substrates that they come across are likely to be naughty in the future. In this issue of Molecular Cell, Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar biochemically reconstitute a key step of a quality control mechanism in the endoplasmic reticulum (ER) that selectively marks terminally misfolded glycoproteins for destruction. Nascent polypeptides enter the ER lumen in an unfolded state, and most receive an asparagine-linked glycan precursor as they translocate across the ER membrane. High luminal concentrations of chaperones and protein disulfide isomerase (PDI) facilitate ER protein folding and the acquisition of native disulfide bonds that stabilize proteins against denaturation. The mechanism of ER-associated degradation (ERAD) of misfolded glycoproteins is best understood in budding yeast and consists of four stages (Buchberger et al., 2010Buchberger A. Bukau B. Sommer T. Mol. Cell. 2010; 40: 238-252Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar): substrate selection in the ER lumen, commitment to degradation, retrotranslocation across the membrane, and ubiquitination and degradation by the cytosolic proteasome (Figure 1). The prevailing model in the field for how misfolded glycoproteins are selected for ERAD is the glycan folding timer. Specifically, as glycoproteins attempt to fold, their sugars are rapidly and sequentially removed, first by glucosidases, and then by the Mnl1 mannosidase, thus yielding a smaller glycan structure with eight mannose residues (Man8). This trimmed glycan is then whittled down further to a specific Man7 structure, in what is thought to be the slowest step that “sets” the timer, to expose an epitope for the sugar-binding domain of the luminal ER lectin Yos9 (Quan et al., 2008Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Lastly, bipartite recognition of the Man7 glycan degron by Yos9 and the unfolded protein substrate by Hrd3, a dedicated ERAD chaperone associated with Yos9, commits misfolded glycoproteins for degradation (Denic et al., 2006Denic V. Quan E.M. Weissman J.S. Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, Gauss et al., 2006Gauss R. Jarosch E. Sommer T. Hirsch C. Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (182) Google Scholar). Thus, the above sugar-trimming mechanism sets a time delay, which ensures that “younger” folding intermediates are spared, while the “older” ones, which have been in the ER long enough to acquire Man7, are efficiently degraded. Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar aimed their study at a critical missing piece in the mechanism of the glycan folding timer model: the identity of the Man8-to-7 mannosidase. Two earlier lines of genetic evidence suggested that Htm1 (Homologous to mannosidase 1) is responsible (Clerc et al., 2009Clerc S. Hirsch C. Oggier D.M. Deprez P. Jakob C. Sommer T. Aebi M. J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (192) Google Scholar). First, overexpression of Htm1 caused enhanced accumulation of cellular Man7. Second, deletion of ALG3, a gene encoding an enzyme required for the biosynthesis of the asparagine-linked glycan precursor, suppressed the ERAD defect of Δhtm1 cells. In this double mutant genetic background, the requirement for Htm1 is bypassed due to protein glycosylation with a glycan biosynthetic intermediate that has a Yos9-binding epitope. To directly examine the mannosidase activity of Htm1, Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar first attempted to express and purify Htm1 using an insect cell baculovirus system but obtained soluble Htm1 in poor yield. Since Htm1 is normally in a stoichiometric complex with PDI (Clerc et al., 2009Clerc S. Hirsch C. Oggier D.M. Deprez P. Jakob C. Sommer T. Aebi M. J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (192) Google Scholar, Sakoh-Nakatogawa et al., 2009Sakoh-Nakatogawa M. Nishikawa S. Endo T. J. Biol. Chem. 2009; 284: 11815-11825Crossref PubMed Scopus (52) Google Scholar), they next coexpressed Htm1 and PDI and successfully purified the Htm1-PDI complex. To measure the mannosidase activity of this complex in vitro, the authors monitored Man8-to-7 conversion in a mixture of total cellular proteins that were denatured, reduced, and alkylated. They found that the Htm1-PDI complex converted ∼10% of the cellular Man8 signal to Man7 under conditions in which enzyme concentration was not limiting. To define a role for PDI in ERAD as part of the Htm1-PDI complex, Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar took advantage of a point mutant allele in the PDI gene (pdi1-1) isolated previously in a genetic screen for mutations that are synthetic lethal with an unfolded protein response (UPR) mutant (Ng et al., 2000Ng D.T. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (266) Google Scholar). The viability of UPR mutants is dependent on normal ERAD function because they are unable to sense unfolded proteins in the ER lumen and transcriptionally upregulate the production of many proteins, including ER chaperones and PDI, which help restore protein homeostasis. The authors go on to provide evidence that oxidative protein folding in pdi1-1 cells is normal, but that Man8-to-7 conversion is reduced. This is consistent with their observation that Pdi1-1 does not efficiently coimmunoprecipitate with Htm1, as well as the enhanced turnover of Htm1 in pdi1-1 cells. The Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar study raises two important questions. First, why is the Htm1 mannosidase activity physically linked to PDI? Part of the answer is that PDI facilitates oxidative folding of Htm1's mannosidase domain by introducing a disulfide bond into it (Sakoh-Nakatogawa et al., 2009Sakoh-Nakatogawa M. Nishikawa S. Endo T. J. Biol. Chem. 2009; 284: 11815-11825Crossref PubMed Scopus (52) Google Scholar). By comparison, homologous mannosidase domains, like the one in Mnl1, also have disulfide bonds, but their biogenesis is not intimately tied to PDI. Intriguingly, the structure of yeast PDI revealed the presence of a defined hydrophobic binding pocket that most likely interacts with unfolded protein substrates (Tian et al., 2006Tian G. Xiang S. Noiva R. Lennarz W.J. Schindelin H. Cell. 2006; 124: 61-73Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Together with the small but significant decrease in Htm1-PDI complex activity toward free Man8 glycan substrates, this observation lead Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar to speculate that PDI affords a folding sensor for the Htm1 mannosidase. This is an interesting hypothesis that should be explored by a more systematic structure/function analysis aimed at generating mutant Htm1-PDI complexes with ERAD-defective PDI alleles that don't interfere with Htm1 biogenesis. Second, why are only ∼10% of denatured Man8-containing glycoproteins trimmed by Htm1-PDI in vitro? Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar argue that this is consistent with the mannosidase/folding sensor model, since not all glycans on canonical ERAD substrates are required to mark them for destruction. For example, of the four glycans on the misfolded point mutant version of carboxypeptidase Y (CPY∗), only the C-terminal one is necessary and sufficient for ERAD. It is difficult, however, to mechanistically compare these in vitro and the in vivo observations for two reasons. First, it is not known if the assumption that Htm1-PDI complex only trims the aforementioned C-terminal glycan is true because a CPY∗-specific mannosidase assay is not available. Perhaps, a genetic way to address this possibility would be to ask if Δalg3 suppresses the degradation defect of the CPY∗ variant carrying just the three non-C-terminal glycans, which is stable in wild-type cells. Second, the precise structural relationship between glycoprotein substrates in a complex cellular mixture that was denatured, reduced, and alkylated in vitro and CPY∗ is less than obvious. Structural details are important, however, because inactive glycan positions can become activated when folding is disrupted nearby (Xie et al., 2009Xie W. Kanehara K. Sayeed A. Ng D.T. Mol. Biol. Cell. 2009; 20: 3317-3329Crossref PubMed Scopus (57) Google Scholar). Thus, the utility of the purified Htm1-PDI complex for dissecting the mechanism of the Man8-to-7 mannosidase reaction will be greatly enhanced by interfacing it with a biochemically defined substrate. Technical advances in conjugating synthetic carbohydrates to model misfolded proteins make this an exciting future prospect. To summarize, the Htm1-PDI complex generates a glycan degron that marks misfolded glycoproteins for destruction. As pointed out in Gauss et al., 2011Gauss R. Kanehara K. Carvalho P. Ng D.T.W. Aebi M. Mol. Cell. 2011; 42 (this issue): 782-793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, the pairing of a general ER folding component with a dedicated piece of ERAD machinery might afford a simple stochastic mechanism, akin to a weighted coin rather than the act of “knowing,” for appropriately partitioning newly synthesized glycoproteins between folding and degradation fates. Specifically, since PDI is much more abundant than Htm1, a misfolded protein is more likely, in any instant, to encounter PDI, which should facilitate folding. If a protein persists in its misfolded state, however, the coin toss will eventually land on the other side, and, at that point, PDI will help Htm1 pull out its cattle gun. A Complex of Pdi1p and the Mannosidase Htm1p Initiates Clearance of Unfolded Glycoproteins from the Endoplasmic ReticulumGauss et al.Molecular CellJune 24, 2011In BriefEndoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man8GlcNAc2 carbohydrates bound to unfolded proteins, yielding Man7GlcNAc2. This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Full-Text PDF Open Archive
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