FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2015086970> ?p ?o ?g. }

• W2015086970 endingPage "591" @default.
• W2015086970 startingPage "579" @default.
• W2015086970 abstract "Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane. Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane. Hrd1p overexpression bypasses the need of other ERAD membrane components Hrd1p oligomerization is required for the degradation of luminal ER substrates A luminal substrate interacts with Hrd1p during retrotranslocation Hrd1p-substrate interaction requires ERAD components and Hrd1p's transmembrane domain Misfolded proteins in the lumen or membrane of the endoplasmic reticulum (ER) are ultimately retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome (for review, see Hirsch et al., 2009Hirsch C. Gauss R. Horn S.C. Neuber O. Sommer T. The ubiquitylation machinery of the endoplasmic reticulum.Nature. 2009; 458: 453-460Crossref PubMed Scopus (272) Google Scholar, Xie and Ng, 2010Xie W. Ng D.T. ERAD substrate recognition in budding yeast.Semin. Cell Dev. Biol. 2010; 21: 533-539Crossref PubMed Scopus (48) Google Scholar). The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. In S. cerevisiae, substrates use three ERAD pathways (ERAD-L, -M, and -C), depending on whether their misfolded domain is located in the ER lumen, in the ER membrane, or on the cytoplasmic side of the ER membrane (Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, Taxis et al., 2003Taxis C. Hitt R. Park S.H. Deak P.M. Kostova Z. Wolf D.H. Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD.J. Biol. Chem. 2003; 278: 35903-35913Crossref PubMed Scopus (160) Google Scholar, Vashist and Ng, 2004Vashist S. Ng D.T.W. Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control.J. Cell Biol. 2004; 165: 41-52Crossref PubMed Scopus (343) Google Scholar). ERAD-L requires a heterotetrameric membrane protein complex, the Hrd1p complex, comprised of the ubiquitin ligase Hrd1p, as well as Hrd3p, Usa1p, and Der1p (Bays et al., 2001aBays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (368) Google Scholar, Bordallo et al., 1998Bordallo J. Plemper R.K. Finger A. Wolf D.H. Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins.Mol. Biol. Cell. 1998; 9: 209-222Crossref PubMed Scopus (284) Google Scholar, Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). ERAD-M requires only a subset of these components. Finally, ERAD-C uses a different ubiquitin ligase (Doa10p) (Swanson et al., 2001Swanson R. Locher M. Hochstrasser M. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (354) Google Scholar). Following polyubiquitination, these pathways converge at an ATPase complex, consisting of the ATPase Cdc48p and two cofactors (Ufd1p and Npl4p) (Bays et al., 2001bBays N.W. Wilhovsky S.K. Goradia A. Hodgkiss-Harlow K. Hampton R.Y. HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins.Mol. Biol. Cell. 2001; 12: 4114-4128Crossref PubMed Scopus (248) Google Scholar, Braun et al., 2002Braun S. Matuschewski K. Rape M. Thoms S. Jentsch S. Role of the ubiquitin-selective CDC48(UFD1/NPL4)chaperone (segregase) in ERAD of OLE1 and other substrates.EMBO J. 2002; 21: 615-621Crossref PubMed Scopus (284) Google Scholar, Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, Jarosch et al., 2002Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48.Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (425) Google Scholar, Rabinovich et al., 2002Rabinovich E. Kerem A. Fröhlich K.U. Diamant N. Bar-Nun S. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation.Mol. Cell. Biol. 2002; 22: 626-634Crossref PubMed Scopus (456) Google Scholar, Ye et al., 2001Ye Y. Meyer H.H. Rapoport T.A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol.Nature. 2001; 414: 652-656Crossref PubMed Scopus (859) Google Scholar). Although most, if not all, components have been identified, the molecular mechanisms of the ERAD pathways remain unclear. Some insight exists into the events that occur during ERAD-L on the luminal and cytoplasmic sides of the ER membrane. Misfolded, glycosylated ERAD-L substrates are initially recognized in the ER lumen. Their prolonged residence time in the ER results in the processing of their carbohydrate moiety to generate a terminal α1,6 mannose residue (Bhamidipati et al., 2005Bhamidipati A. Denic V. Quan E.M. Weissman J.S. Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen.Mol. Cell. 2005; 19: 741-751Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, Clerc et al., 2009Clerc S. Hirsch C. Oggier D.M. Deprez P. Jakob C. Sommer T. Aebi M. Htm1 protein generates the N-glycan signal for glycoprotein degradation in the endoplasmic reticulum.J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (192) Google Scholar, Kim et al., 2005Kim W. Spear E.D. Ng D.T. Yos9p detects and targets misfolded glycoproteins for ER-associated degradation.Mol. Cell. 2005; 19: 753-764Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, Quan et al., 2008Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Defining the glycan destruction signal for endoplasmic reticulum-associated degradation.Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, Szathmary et al., 2005Szathmary R. Bielmann R. Nita-Lazar M. Burda P. Jakob C.A. Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD.Mol. Cell. 2005; 19: 765-775Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This sugar, together with the unfolded polypeptide segment surrounding the carbohydrate attachment site, constitutes the degradation signal (Xie et al., 2009Xie W. Kanehara K. Sayeed A. Ng D.T. Intrinsic conformational determinants signal protein misfolding to the Hrd1/Htm1 endoplasmic reticulum-associated degradation system.Mol. Biol. Cell. 2009; 20: 3317-3329Crossref PubMed Scopus (57) Google Scholar). The signal is then recognized through a luminal domain of Hrd3p as well as the lectin Yos9p (Denic et al., 2006Denic V. Quan E.M. Weissman J.S. A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, Gauss et al., 2006aGauss R. Jarosch E. Sommer T. Hirsch C. A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery.Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (182) Google Scholar). Once the substrate appears on the cytoplasmic side of the ER membrane, the RING finger domain of Hrd1p, together with the ubiquitin-conjugating enzymes Ubc7p or Ubc1p, catalyze polyubiquitination (Bays et al., 2001aBays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (368) Google Scholar, Bordallo et al., 1998Bordallo J. Plemper R.K. Finger A. Wolf D.H. Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins.Mol. Biol. Cell. 1998; 9: 209-222Crossref PubMed Scopus (284) Google Scholar). The modified substrate is then recognized by the Cdc48p/Ufd1p/Npl4p ATPase complex and moved into the cytosol (Bays et al., 2001bBays N.W. Wilhovsky S.K. Goradia A. Hodgkiss-Harlow K. Hampton R.Y. HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins.Mol. Biol. Cell. 2001; 12: 4114-4128Crossref PubMed Scopus (248) Google Scholar, Braun et al., 2002Braun S. Matuschewski K. Rape M. Thoms S. Jentsch S. Role of the ubiquitin-selective CDC48(UFD1/NPL4)chaperone (segregase) in ERAD of OLE1 and other substrates.EMBO J. 2002; 21: 615-621Crossref PubMed Scopus (284) Google Scholar, Jarosch et al., 2002Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48.Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (425) Google Scholar, Rabinovich et al., 2002Rabinovich E. Kerem A. Fröhlich K.U. Diamant N. Bar-Nun S. AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation.Mol. Cell. Biol. 2002; 22: 626-634Crossref PubMed Scopus (456) Google Scholar, Ye et al., 2001Ye Y. Meyer H.H. Rapoport T.A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol.Nature. 2001; 414: 652-656Crossref PubMed Scopus (859) Google Scholar). Finally, the substrate is delivered to the proteasome and degraded. The events of ERAD-L occurring inside the ER membrane are unknown, particularly the mechanism by which substrates are moved through the ER membrane. It has been proposed that a retrotranslocation channel is required, and several channel candidates have been considered (for review, see Hirsch et al., 2009Hirsch C. Gauss R. Horn S.C. Neuber O. Sommer T. The ubiquitylation machinery of the endoplasmic reticulum.Nature. 2009; 458: 453-460Crossref PubMed Scopus (272) Google Scholar). Among the components of the Hrd1p complex, the most attractive candidates are Der1p and its mammalian homologs, the Derlins (Knop et al., 1996Knop M. Finger A. Braun T. Hellmuth K. Wolf D.H. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.EMBO J. 1996; 15: 753-763Crossref PubMed Scopus (309) Google Scholar, Lilley and Ploegh, 2004Lilley B.N. Ploegh H.L. A membrane protein required for dislocation of misfolded proteins from the ER.Nature. 2004; 429: 834-840Crossref PubMed Scopus (559) Google Scholar, Ye et al., 2004Ye Y. Shibata Y. Yun C. Ron D. Rapoport T.A. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol.Nature. 2004; 429: 841-847Crossref PubMed Scopus (764) Google Scholar), as well as Hrd1p, simply because they possess the largest number of transmembrane segments. In addition, some studies suggest a role for the Sec61 channel, which is normally involved in the translocation of proteins from the cytosol into the ER (Pilon et al., 1997Pilon M. Schekman R. Römisch K. Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.EMBO J. 1997; 16: 4540-4548Crossref PubMed Scopus (338) Google Scholar, Schäfer and Wolf, 2009Schäfer A. Wolf D.H. Sec61p is part of the endoplasmic reticulum-associated degradation machinery.EMBO J. 2009; 28: 2874-2884Crossref PubMed Scopus (60) Google Scholar, Wiertz et al., 1996Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.Nature. 1996; 384: 432-438Crossref PubMed Scopus (936) Google Scholar, Willer et al., 2008Willer M. Forte G.M. Stirling C.J. Sec61p is required for ERAD-L: genetic dissection of the translocation and ERAD-L functions of Sec61P using novel derivatives of CPY.J. Biol. Chem. 2008; 283: 33883-33888Crossref PubMed Scopus (66) Google Scholar, Zhou and Schekman, 1999Zhou M. Schekman R. The engagement of Sec61p in the ER dislocation process.Mol. Cell. 1999; 4: 925-934Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, there is no direct evidence in support of any of these candidates, and it is not even clear whether a channel exists. In fact, it has been suggested that translocation is instead linked to the formation of lipid droplets (Ploegh, 2007Ploegh H.L. A lipid-based model for the creation of an escape hatch from the endoplasmic reticulum.Nature. 2007; 448: 435-438Crossref PubMed Scopus (213) Google Scholar). How the driving force for retrotranslocation is provided is also unclear. Whereas it is conceivable that the Cdc48p ATPase pulls on a polyubiquitinated substrate once it appears on the cytosolic side of the membrane, it is mysterious how energy would be provided for moving the polypeptide through the membrane to make it accessible to the ubiquitination machinery. Finally, and more generally, the specific functions of the individual components of the Hrd1p-complex are unknown, and it is unclear whether they act in a temporal order during retrotranslocation of ERAD-L substrates. Here, we show that Hrd1p is the central membrane component in the ERAD-L process and propose a model for polypeptide movement through the ER membrane. In wild-type S. cerevisiae cells, all four components of the Hrd1p complex (Hrd1p, Hrd3p, Usa1p, and Der1p) are essential for the degradation of ERAD-L substrates (Knop et al., 1996Knop M. Finger A. Braun T. Hellmuth K. Wolf D.H. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.EMBO J. 1996; 15: 753-763Crossref PubMed Scopus (309) Google Scholar, Taxis et al., 2003Taxis C. Hitt R. Park S.H. Deak P.M. Kostova Z. Wolf D.H. Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD.J. Biol. Chem. 2003; 278: 35903-35913Crossref PubMed Scopus (160) Google Scholar, Gauss et al., 2006aGauss R. Jarosch E. Sommer T. Hirsch C. A complex of Yos9p and the HRD ligase integrates endoplasmic reticulum quality control into the degradation machinery.Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (182) Google Scholar, Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). However, we reasoned that the overexpression of one component may make other components dispensable, a result that would indicate a functional hierarchy among these factors. These experiments were also motivated by the previous observation that overexpression of Hrd1p compensates for the absence of Hrd3p (Gardner et al., 2000Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p.J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (234) Google Scholar, Plemper et al., 1999Plemper R.K. Bordallo J. Deak P.M. Taxis C. Hitt R. Wolf D.H. Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation.J. Cell Sci. 1999; 112: 4123-4134Crossref PubMed Google Scholar), although this result was interpreted as simply restoring the levels of Hrd1p, which becomes unstable in a HRD3 deletion mutant. We tested the effect of overexpression of Hrd1p on the degradation of a well-characterized ERAD-L substrate, a misfolded version of carboxypeptidase Y (CPY∗) that is tagged with a C-terminal hemagglutinin (HA) epitope (Finger et al., 1993Finger A. Knop M. Wolf D.H. Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast.Eur. J. Biochem. 1993; 218: 565-574Crossref PubMed Scopus (168) Google Scholar, Ng et al., 2000Ng D.T. Spear E.D. Walter P. The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control.J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (267) Google Scholar). In wild-type cells, CPY∗-HA is degraded with a half-life of ∼30 min (Figure 1A , lanes 1–4 and graph). When the endogenous promoter for Hrd1p was replaced by the strong, galactose-inducible GAL1 promoter and the cells were grown in the presence of galactose, CPY∗-HA degradation was accelerated (Figure 1A, lanes 5–8). In the presence of glucose, when the Gal promoter was repressed, CPY∗-HA was stable, as expected from the depletion of Hrd1p (Figure S1A available online). Although overexpressed Hrd1p is unstable, the steady-state levels are increased by a factor of ∼10 (Figure S1B). CPY∗-HA degradation by overexpressed Hrd1p was much slower when the ubiquitin-conjugating enzyme Ubc7p was absent (Figure 1A, lanes 9–12) and was completely abrogated when an essential cysteine in the RING finger domain of Hrd1p was mutated (Figure S1C). In addition, degradation was attenuated in a cdc48 mutant (Figure S1C). Thus, the requirements for ubiquitin-ligase activity by Hrd1p and for the function of the Cdc48 ATPase are maintained when Hrd1p is overexpressed.Figure S1Stability of CPY∗ and Hrd1p in Cells with the Indicated Genotype Expressing Different Levels of Hrd1p, Related to Figure 1Show full caption(A) The degradation of the misfolded luminal ER protein CPY∗-HA was followed after inhibition of protein synthesis by cycloheximide in wild-type (wt) cells or in cells in which Hrd1p was placed under the control of the GAL promoter. Hrd1p expression was repressed in presence of glucose (noninducing conditions). Where indicated, genes for ERAD components were deleted.(B) The levels and stability of Hrd1p were analyzed in wt cells or in cells overexpressing Hrd1p from the GAL1 promoter in the presence of galactose. Where indicated, genes for ERAD components were deleted.(C) As in (A) but in hrd1Δ cells overexpressing wild-type Hrd1p or a Hrd1p mutant defective in its ubiquitin ligase activity (C399S). Where indicated, the cells expressed a temperature-sensitive mutation in CDC48 (cdc48-3) and were analyzed after 2 hr incubation at the restrictive temperature of 37°C.(D) As in (A) but in hrd1Δ hrd3Δusa1Δ der1Δ cells overexpressing either wild-type Hrd1p or a ubiquitin-ligase-deficient mutant (C399S) under the GAL promoter in the presence of galactose (top). The bottom panel shows the levels of wild-type and mutant Hrd1p in the same experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The degradation of the misfolded luminal ER protein CPY∗-HA was followed after inhibition of protein synthesis by cycloheximide in wild-type (wt) cells or in cells in which Hrd1p was placed under the control of the GAL promoter. Hrd1p expression was repressed in presence of glucose (noninducing conditions). Where indicated, genes for ERAD components were deleted. (B) The levels and stability of Hrd1p were analyzed in wt cells or in cells overexpressing Hrd1p from the GAL1 promoter in the presence of galactose. Where indicated, genes for ERAD components were deleted. (C) As in (A) but in hrd1Δ cells overexpressing wild-type Hrd1p or a Hrd1p mutant defective in its ubiquitin ligase activity (C399S). Where indicated, the cells expressed a temperature-sensitive mutation in CDC48 (cdc48-3) and were analyzed after 2 hr incubation at the restrictive temperature of 37°C. (D) As in (A) but in hrd1Δ hrd3Δusa1Δ der1Δ cells overexpressing either wild-type Hrd1p or a ubiquitin-ligase-deficient mutant (C399S) under the GAL promoter in the presence of galactose (top). The bottom panel shows the levels of wild-type and mutant Hrd1p in the same experiment. Next, we tested CPY∗-HA degradation in cells that overexpressed Hrd1p but lacked other components of the Hrd1p complex. As reported (Plemper et al., 1999Plemper R.K. Bordallo J. Deak P.M. Taxis C. Hitt R. Wolf D.H. Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation.J. Cell Sci. 1999; 112: 4123-4134Crossref PubMed Google Scholar), degradation was not affected when Hrd3p was absent (Figure 1A, lanes 13–16). Of interest, the process was also not affected by the absence of Usa1p or Der1p (Figure 1A, lanes 17–20 and 21–24). Moreover, even in cells that simultaneously lacked Hrd3p, Usa1p, and Der1p, the degradation of CPY∗-HA was unimpeded (Figure 1A, lanes 25–28). Degradation in the triple-deletion mutant required Hrd1p expression (Figure S1A) and ubiquitin ligase activity (Figure S1D). These experiments indicate that Hrd1p overexpression bypasses the need for the other components of the Hrd1p complex. Similar results were obtained with KHN-HA (a soluble protein) and KWW-HA (a membrane-bound protein), both containing a misfolded luminal domain (Figure S2) (Vashist and Ng, 2004Vashist S. Ng D.T.W. Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control.J. Cell Biol. 2004; 165: 41-52Crossref PubMed Scopus (343) Google Scholar). The bypass effect observed with Hrd1p was not seen when the other components of the Hrd1p complex were overexpressed. Der1p under the Gal promoter did not accelerate CPY∗-HA degradation and did not alleviate the requirement for the other Hrd1p complex components (Figure 1B). Usa1p or Hrd3p overexpression blocked degradation of CPY∗-HA (Figures 1C and 1D), indicating that excess of these components interferes with the normal function of the Hrd1p complex. Taken together, our data suggest that Hrd1p is the key component of the Hrd1p complex and that the other subunits may have ancillary roles. We hypothesized that Hrd1p overexpression bypasses the need of the other Hrd1p complex components because Hrd1p must oligomerize to be active in ERAD-L; in wild-type cells, the oligomerization would be regulated by other components of the Hrd1p complex, whereas Hrd1p overexpression would force its spontaneous oligomerization. Both our own previous experiments and more recent results indicate that endogenous Hrd1p forms high-molecular weight complexes whose size depends on the presence of Usa1p, but not Hrd3p or Der1p (Carvalho et al., 2006Carvalho P. Goder V. Rapoport T.A. Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, Horn et al., 2009Horn S.C. Hanna J. Hirsch C. Volkwein C. Schütz A. Heinemann U. Sommer T. Jarosch E. Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol. Cell. 2009; 36: 782-793Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To investigate directly the oligomerization of Hrd1p, we expressed Myc- and HA-tagged versions of Hrd1p in the same cell, both under the endogenous promoter. Detergent-solubilized membrane extracts were then treated with bifunctional crosslinking reagents and subjected to immunoprecipitation with HA antibodies. With extracts from wild-type cells that were not treated with crosslinker, HA antibodies precipitated Hrd1p-Myc (Figure 2A , top panel, lane 4), as previously described (Horn et al., 2009Horn S.C. Hanna J. Hirsch C. Volkwein C. Schütz A. Heinemann U. Sommer T. Jarosch E. Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol. Cell. 2009; 36: 782-793Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Upon treatment with crosslinkers, several high-molecular weight bands were observed (Figure 2A, top panel, lanes 5 and 6). The highest molecular weight band appeared with multiple crosslinkers and contains not only Hrd1p, but also Usa1p (Figure 2A, bottom panel, lanes 5 and 6). With extracts lacking Usa1p, HA antibodies precipitated only negligible amounts of Hrd1p-Myc or the crosslinked bands (Figure 2A, bottom panel, lanes 1–3). Thus, Usa1p facilitates Hrd1p oligomerization. Next, we identified the regions in Usa1p that are responsible for Hrd1p interaction and oligomerization. Usa1p contains two transmembrane segments flanked by long cytoplasmic domains (Figure 2B). Preliminary experiments showed that the cytoplasmic region preceding the first transmembrane segment was required for both Hrd1p oligomerization and Usa1p interaction (data not shown). For a more precise analysis, we introduced deletions into Usa1p (Figure 2B). We found a segment (437–490), called segment H, whose deletion abolished both the binding of Usa1p to Hrd1p and the oligomerization of Hrd1p (Figure 2B, lane 12). Segment H likely interacts with the C-terminal 34 residues of Hrd1p (Horn et al., 2009Horn S.C. Hanna J. Hirsch C. Volkwein C. Schütz A. Heinemann U. Sommer T. Jarosch E. Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol. Cell. 2009; 36: 782-793Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Another deletion (residues 319–418) left the interaction between Usa1p and Hrd1p intact but significantly reduced Hrd1p oligomerization (Figure 2B, lane 10). The region from 371 to 418, called segment U, appears to be important for Hrd1p oligomerization because a deletion mutant lacking residues 319–371 behaves like wild-type Usa1p (Figure 2B, lane 9). A recent report suggested that the segment from 259 to 312 encompassing the Ubl domain is responsible for inducing oligomers of Hrd1p (Horn et al., 2009Horn S.C. Hanna J. Hirsch C. Volkwein C. Schütz A. Heinemann U. Sommer T. Jarosch E. Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol. Cell. 2009; 36: 782-793Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). However, we did not observe any defects of Hrd1p oligomerization upon complete deletion of the Ubl domain (259–318) (Figure S3A ). A simple model for Usa1p-dependent Hrd1p oligomerization is that Usa1p itself forms oligomers through segment U and binds Hrd1p through segment H (see scheme in Figure 7A). To test this idea, we expressed HA-tagged full-length Usa1p together with FLAG-tagged versions of Usa1p mutants, all under the endogenous promoter. HA antibodies precipitated all Usa1p-FLAG constructs, with the exception of the one lacking segment U (Figure 2C). Similar results were obtained with hrd1Δ cells (data not shown). These results support a model in which Usa1p oligomers facilitate Hrd1p oligomerization. Based on our overexpression experiments, it appears that Hrd1p has an intrinsic propensity to form oligomers, which are stabilized by Usa1p. Indeed, crosslinking experiments show that overexpressed Hrd1p can form high molecular weight species even in the absence of Usa1p (Figure S3B). This is further supported by experiments in which ERAD was inactivated by a mutation of an essential cysteine in the RING finger of Hrd1p (C399S). As expected (Horn et al., 2009Horn S.C. Hanna J. Hirsch C. Volkwein C. Schütz A. Heinemann U. Sommer T. Jarosch E. Usa1 functions as a scaffold of the HRD-ubiquitin ligase.Mol. Cell. 2009; 36: 782-793Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), in the presence of Usa1p, this mutant protein formed oligomers (Figure 2D, lane 11). Of interest, however, Hrd1p (C399S) oligomerized even in the absence of Usa1p (Figure 2D, lanes 15" @default.
• W2015086970 created "2016-06-24" @default.
• W2015086970 creator A5002573490 @default.
• W2015086970 creator A5077706232 @default.
• W2015086970 creator A5087757787 @default.
• W2015086970 date "2010-11-01" @default.
• W2015086970 modified "2023-10-12" @default.
• W2015086970 title "Retrotranslocation of a Misfolded Luminal ER Protein by the Ubiquitin-Ligase Hrd1p" @default.
• W2015086970 cites W1668688100 @default.
• W2015086970 cites W1965839025 @default.
• W2015086970 cites W1966072209 @default.
• W2015086970 cites W1976601347 @default.
• W2015086970 cites W1981625315 @default.
• W2015086970 cites W1981835453 @default.
• W2015086970 cites W1983463562 @default.
• W2015086970 cites W1984347348 @default.
• W2015086970 cites W1984354326 @default.
• W2015086970 cites W1986958303 @default.
• W2015086970 cites W1996109826 @default.
• W2015086970 cites W2000447975 @default.
• W2015086970 cites W2000456081 @default.
• W2015086970 cites W2010478265 @default.
• W2015086970 cites W2015519581 @default.
• W2015086970 cites W2017776769 @default.
• W2015086970 cites W2018515506 @default.
• W2015086970 cites W2025226894 @default.
• W2015086970 cites W2028997060 @default.
• W2015086970 cites W2030723839 @default.
• W2015086970 cites W2031488588 @default.
• W2015086970 cites W2033088972 @default.
• W2015086970 cites W2034133901 @default.
• W2015086970 cites W2034289224 @default.
• W2015086970 cites W2035535713 @default.
• W2015086970 cites W2042447745 @default.
• W2015086970 cites W2043079553 @default.
• W2015086970 cites W2049127225 @default.
• W2015086970 cites W2049656093 @default.
• W2015086970 cites W2051881845 @default.
• W2015086970 cites W2060391020 @default.
• W2015086970 cites W2064609154 @default.
• W2015086970 cites W2065600580 @default.
• W2015086970 cites W2071046609 @default.
• W2015086970 cites W2080928193 @default.
• W2015086970 cites W2114953435 @default.
• W2015086970 cites W2115564529 @default.
• W2015086970 cites W2118278390 @default.
• W2015086970 cites W2122551230 @default.
• W2015086970 cites W2130368151 @default.
• W2015086970 cites W2139577310 @default.
• W2015086970 cites W2145174769 @default.
• W2015086970 cites W2145922063 @default.
• W2015086970 cites W2146183177 @default.
• W2015086970 cites W2146969978 @default.
• W2015086970 cites W2152726884 @default.
• W2015086970 cites W2155946179 @default.
• W2015086970 cites W2169221124 @default.
• W2015086970 doi "https://doi.org/10.1016/j.cell.2010.10.028" @default.
• W2015086970 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3026631" @default.
• W2015086970 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21074049" @default.
• W2015086970 hasPublicationYear "2010" @default.
• W2015086970 type Work @default.
• W2015086970 sameAs 2015086970 @default.
• W2015086970 citedByCount "265" @default.
• W2015086970 countsByYear W20150869702012 @default.
• W2015086970 countsByYear W20150869702013 @default.
• W2015086970 countsByYear W20150869702014 @default.
• W2015086970 countsByYear W20150869702015 @default.
• W2015086970 countsByYear W20150869702016 @default.
• W2015086970 countsByYear W20150869702017 @default.
• W2015086970 countsByYear W20150869702018 @default.
• W2015086970 countsByYear W20150869702019 @default.
• W2015086970 countsByYear W20150869702020 @default.
• W2015086970 countsByYear W20150869702021 @default.
• W2015086970 countsByYear W20150869702022 @default.
• W2015086970 countsByYear W20150869702023 @default.
• W2015086970 crossrefType "journal-article" @default.
• W2015086970 hasAuthorship W2015086970A5002573490 @default.
• W2015086970 hasAuthorship W2015086970A5077706232 @default.
• W2015086970 hasAuthorship W2015086970A5087757787 @default.
• W2015086970 hasBestOaLocation W20150869701 @default.
• W2015086970 hasConcept C104317684 @default.
• W2015086970 hasConcept C134459356 @default.
• W2015086970 hasConcept C204328495 @default.
• W2015086970 hasConcept C25602115 @default.
• W2015086970 hasConcept C2908868211 @default.
• W2015086970 hasConcept C54355233 @default.
• W2015086970 hasConcept C86803240 @default.
• W2015086970 hasConcept C95444343 @default.
• W2015086970 hasConceptScore W2015086970C104317684 @default.
• W2015086970 hasConceptScore W2015086970C134459356 @default.
• W2015086970 hasConceptScore W2015086970C204328495 @default.
• W2015086970 hasConceptScore W2015086970C25602115 @default.
• W2015086970 hasConceptScore W2015086970C2908868211 @default.
• W2015086970 hasConceptScore W2015086970C54355233 @default.
• W2015086970 hasConceptScore W2015086970C86803240 @default.
• W2015086970 hasConceptScore W2015086970C95444343 @default.
• W2015086970 hasIssue "4" @default.
• W2015086970 hasLocation W20150869701 @default.

• next