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• W1994485108 abstract "Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, ΔssCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of ΔssCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD. Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, ΔssCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of ΔssCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD. Misfolded proteins and orphan subunits of oligomeric proteins may account for up to 30% of newly synthesized proteins (1Schubert U. Anton L.C. Gibbs J. Norbury C.C. Yewdell J.W. Bennink J.R. Nature. 2000; 404: 770-774Crossref PubMed Scopus (1) Google Scholar). Such aberrant proteins are deleterious to the cell and therefore must be eliminated (2Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3352) Google Scholar). In the endoplasmic reticulum (ER), 4The abbreviations used are:ERendoplasmic reticulumCPcatalytic particleERADER-associated protein degradationRPregulatory particleUPRunfolded protein responseHAhemagglutininGFPgreen fluorescent proteinX-gal5-bromo-4-chloro-3-indolyl-β-d-galactoside. quality control mechanisms assure that aberrant proteins are not exported; instead, they are usually dislocated to the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER-associated protein degradation (ERAD; for reviews see Refs. 3Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar, 4Lederkremer G.Z. Glickman M.H. Trends Biochem. Sci. 2005; 30: 297-303Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 5Bar-Nun S. Curr. Top. Microbiol. Immunol. 2005; 300: 95-125PubMed Google Scholar). Once selected for ERAD, both membrane and luminal proteins are dislocated from the ER back to the cytosol, tagged by polyubiquitin, and delivered for their irreversible proteolysis by the 26S proteasome (6Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (616) Google Scholar). endoplasmic reticulum catalytic particle ER-associated protein degradation regulatory particle unfolded protein response hemagglutinin green fluorescent protein 5-bromo-4-chloro-3-indolyl-β-d-galactoside. The cytosolic p97/Cdc48 ATPase complex appears to provide the driving force for the dislocation of ERAD substrates. This homo-hexameric AAA-ATPase along with its cofactors Ufd1 and Npl4 was shown to be essential for ERAD (7Bays N.W. Wilhovsky S.K. Goradia A. Hodgkiss-Harlow K. Hampton R.Y. Mol. Biol. Cell. 2001; 12: 4114-4128Crossref PubMed Scopus (258) Google Scholar, 8Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Crossref PubMed Scopus (900) Google Scholar, 9Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell Biol. 2002; 22: 626-634Crossref PubMed Scopus (471) Google Scholar, 10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar, 11Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol. 2003; 162: 71-84Crossref PubMed Scopus (501) Google Scholar, 12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Involvement of the p97/Cdc48 complex in the dislocation step was demonstrated by the inhibiting effect of p97 dominant negative variant on dislocation of MHC class I heavy chain in semiintact mammalian cells (8Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Crossref PubMed Scopus (900) Google Scholar) and by the hampered release of CPY* to the cytosol in the ufd1-1 mutant (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar). In the cdc48-10 mutant, CPY* remained trapped within the ER lumen as non-ubiquitinated protein, pointing to Cdc48 as the driving force for passage across ER membranes (12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The ability of p97/Cdc48 to pull ERAD substrates out of the ER is probably a consequence of conformational changes of p97 during its ATPase cycle, which translate ATP hydrolysis into mechanical forces (13Rouiller I. DeLaBarre B. May A.P. Weis W.I. Brunger A.T. Milligan R.A. Wilson-Kubalek E.M. Nat. Struct. Biol. 2002; 9: 950-957Crossref PubMed Scopus (184) Google Scholar), and the underlying activity of AAA-ATPases in unfolding and disassembling proteins (14Lupas A.N. Martin J. Curr. Opin. Struct. Biol. 2002; 12: 746-753Crossref PubMed Scopus (298) Google Scholar). An alternative candidate to provide the driving force for dislocation of ERAD substrates is the proteasome. Being a limiting factor in the entire ubiquitin-dependent proteolytic pathway, proteasomes greatly influence system capacity and the substrates degradation rate, including ERAD (15Bajorek M. Finley D. Glickman M.H. Curr. Biol. 2003; 13: 1140-1144Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Rabinovich E. Bajorek M. Glickman M. Bar-Nun S. Israel J. Chem. 2006; 46: 219-224Crossref Scopus (5) Google Scholar). Yet, in addition to its established function in proteolysis at the end step of the pathway, the 26S proteasome may also participate in earlier steps, such as dislocation of ERAD substrates. Mechanistically, proteasome association with ER membranes would facilitate its function in pulling substrates out of the ER. Indeed, a proteasome subpopulation is associated with the ER (for reviews see Refs. 17Rivett A.J. Curr. Opin. Immunol. 1998; 10: 110-114Crossref PubMed Scopus (142) Google Scholar, 18Hirsch C. Ploegh H.L. Trends Cell Biol. 2000; 10: 268-272Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The two subcomplexes of the 26S proteasome, the 20S catalytic particle (CP) and the ATPase-containing 19S regulatory particle (RP), were implicated in dislocation. The proteolytic activity of β-subunits within the 20S CP was shown to be required for the extraction of several ERAD substrates from the ER (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar, 19Mayer T.U. Braun T. Jentsch S. EMBO J. 1998; 17: 3251-3257Crossref PubMed Scopus (179) Google Scholar). Nevertheless, the proteolytic activity of the proteasome does not constitute a general pulling mechanism, as exemplified by ERAD of luminal μs, the heavy chain of secretory IgM, in B lymphocytes (12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Elkabetz Y. Kerem A. Tencer L. Winitz D. Kopito R.R. Bar-Nun S. J. Biol. Chem. 2003; 278: 18922-18929Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Although μs interacted exclusively with ER-bound proteasomes, blockers of proteasomal proteolytic activity had no effect on passage of μs across ER membranes, yet they inhibited subsequent μs release from the ER cytosolic face (12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The 19S RP was also implicated in dislocation and was proposed as the sole driving force for dislocation of pro-alpha factor in vitro (21Lee R.J. Liu C.W. Harty C. McCracken A.A. Latterich M. Romisch K. DeMartino G.N. Thomas P.J. Brodsky J.L. EMBO J. 2004; 23: 2206-2215Crossref PubMed Scopus (98) Google Scholar). The 19S RP is composed of a base subassembly that contains six AAA-ATPase subunits (Rpt1-6) alongside three non-ATPase subunits (Rpn1, Rpn2, Rpn10) and a lid sub-particle that encompasses eight stoichiometric subunits (Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, and Rpn12) (22DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chuping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 23Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell Biol. 1998; 18: 3149-3162Crossref PubMed Google Scholar, 24Verma R. Chen S. Feldman R. Schieltz D. Yates J. Dohmen J. Deshaies R.J. Mol. Biol. Cell. 2000; 11: 3425-3439Crossref PubMed Scopus (461) Google Scholar). The chaperone-like activity of the Rpt AAA-ATPases (25Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.M. Finley D. Schmidt M. Nat. Cell Biology. 1999; 1: 221-226Crossref PubMed Scopus (388) Google Scholar) is crucial for their ability to unfold protein substrates and translocate them through the gated channel into the 20S CP proteolytic chamber. Binding of the 19S RP to the surface of the 20S CP opens the narrow entrance gated by the N-terminal tails of the α-subunits and allows access of protein substrates into the proteolytic chamber for proteolysis (26Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (657) Google Scholar, 27Kohler A. Bajorek M. Groll M. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Biochimie (Paris). 2001; 83: 325-332Crossref PubMed Scopus (64) Google Scholar, 28Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In addition, the Rpt subunits may be involved in binding or anchoring polyubiquitinated substrates since at least one of them, Rpt5/S6, interacts with polyubiquitin chains (29Lam Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Crossref PubMed Scopus (366) Google Scholar). Whether each Rpt AAA-ATPase contributes to ERAD differentially is not clear, and the possibility that distinct sets of Rpt subunits participate preferentially in ERAD or cytosolic/nuclear degradation has been partially explored. Studies with yeast mutants such as cim5-1(rpt1), rpt1S, rpt2RF, rpt4R, rpt5S and cim3-1(rpt6) demonstrated involvement of Rpt subunits in ERAD (6Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (616) Google Scholar, 10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar, 19Mayer T.U. Braun T. Jentsch S. EMBO J. 1998; 17: 3251-3257Crossref PubMed Scopus (179) Google Scholar, 30Hill K. Cooper A.A. EMBO J. 2000; 19: 550-561Crossref PubMed Scopus (93) Google Scholar). Subunit Cim5/Rpt1 was involved in extracting membrane ERAD substrates (19Mayer T.U. Braun T. Jentsch S. EMBO J. 1998; 17: 3251-3257Crossref PubMed Scopus (179) Google Scholar), and subunits Rpt4 and Rpt5, but not Rpt2, were also implicated in ERAD (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar). The luminal ERAD substrate CPY* was stabilized in the ATPase mutants rpt4R and rpt5S but hardly in rpt2RF (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar). However, a small but significant proportion of CPY* remained protease-sensitive in rpt4R, suggesting that protein dislocation could occur independently of Rpt4 ATPase (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar). Hence, it was concluded that p97/Cdc48/Ufd1/Npl4 complex rather than the Rpt subunits provided the driving force for extracting ERAD substrates from the ER (10Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Crossref PubMed Scopus (440) Google Scholar). Intrigued by the possibility that distinct sets of proteasomal AAA-ATPases might be engaged in unique processes along the ERAD pathway, we systematically studied individual contributions of the ATPase activity of each of the six 19S AAA-ATPases to proteasomal degradation globally and to ERAD and dislocation of ERAD substrates in particular. To that end, we utilized a set of six mutant strains in which each RPT gene was replaced by an equivalent mutant version with conservative substitutions of the invariant lysine of the Walker A ATP-binding motif (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). This substitution generally results in complete or partial inhibition of ATP binding and ATP hydrolysis (32Sung P. Higgins D. Prakash L. Prakash S. EMBO J. 1988; 7: 3263-3269Crossref PubMed Scopus (221) Google Scholar). This set of strains allows us to elucidate roles of individual Rpt subunits in the various ERAD steps and to study their interrelation with another AAA-ATPase implicated in ERAD, p97/Cdc48, in fulfilling their multiple functions in ERAD. We found that out of the six equivalent mutated Rpt subunits, only rpt2RF and rpt4R displayed impaired ERAD. However, while the Rpt2 mutation disrupted general proteasomal degradation, the Rpt4 mutation was manifested in impaired dislocation of ERAD substrates, an outcome that was partially restored by excess Cdc48. 5Some preliminary results relating to this article were presented in a Symposium on Ubiquitin in Biology and Medicine, in honor of Nobel Laureates Avram Hersko and Aaron Ciechanover, Israel Academy of Sciences and Humanities, October 31, 2005. This lecture was published in Ref. 60Rabinovich E. Bussi D. Shapira I. Alalouf G. Lipson C. Elkabetz Y. Glickman M. Bajorek M. Bar-Nun. S. Isr. Med. Assoc. J. 2006; 8: 238-242PubMed Google Scholar. Yeast Strains, Media, and Plasmids—Strain SUB62 (MATa his3-D200 lys2-801 leu2-3,112 trp1-1 ura3-52) is the isogenic wild-type of the equivalent rpt mutant strains rpt1S DY106, rpt2RF DY62, rpt3R DY93, rpt4R DY219, rpt5R DY155, and rpt6R DY100, individually mutated in their invariant ATP-binding lysine (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). Defective ERAD was previously shown in cdc48-10 at 37 °C (9Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell Biol. 2002; 22: 626-634Crossref PubMed Scopus (471) Google Scholar). The pre1-1 strain harbors a proteasomal β-subunit defective in chymotrypsin-like activity at 37 °C (33Heinemeyer W. Kleinschmidt J.A. Saidowsky J. Escher C. Wolf D.H. EMBO J. 1991; 10: 555-562Crossref PubMed Scopus (358) Google Scholar). CPY*-HA (prc1-1 allele; plasmid pBG15) and 6myc-Hmg2 (plasmid pRH244) were previously described (9Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell Biol. 2002; 22: 626-634Crossref PubMed Scopus (471) Google Scholar, 12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). ΔssCPY*-GFP was expressed from plasmid POW0668 ((34Medicherla B. Kostova Z. Schaefer A. Wolf D.H. EMBO Rep. 2004; 5: 692-697Crossref PubMed Scopus (170) Google Scholar); generously provided by D. Wolf, Stuttgart University). To replace LEU2 with TRP1 selection marker in Cdc48 expressing plasmid, the CDC48 gene was excised from pKF700 plasmid (generously provided by K. U. Fröhlich, Graz University) by HindIII, bluntended and inserted into a SmaI site in the 2-μm ori-type pAMT20 shuttle vector. Growth Sensitivity to Tunicamycin, Cadmium, Canavanine, and Temperature—Yeast strains grown to equal cell density were spotted on plates as 10-fold serial dilutions. Deficiencies in the ubiquitin-proteasome pathway were monitored either as a temperature-sensitive phenotype or on arginine-deficient plates supplemented with the arginine analog, canavanine (3 μg/ml) (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar, 35Fu H. Reis N. Lee Y. Glickman M.H. Vierstra R.D. EMBO J. 2001; 20: 7096-7107Crossref PubMed Scopus (210) Google Scholar). Deficiencies in ERAD were monitored on plates supplemented with CdCl2 (30 μm) (36Jungmann J. Reins H.A. Schobert C. Jentsch S. Nature. 1993; 361: 369-371Crossref PubMed Scopus (235) Google Scholar, 37Wang Q. Chang A. EMBO J. 2003; 22: 3792-3802Crossref PubMed Scopus (52) Google Scholar) or with tunicamycin (2 μg/ml) (38Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar). Unfolded Protein Response (UPR) Activation—UPR activation was measured with the UPRE-lacZ reporter construct (39Zhou M. Schekman R. Mol. Cell. 1999; 4: 925-934Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Activity of β-galactosidase was detected on 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) reporter plates. The maximal induction of UPR was achieved with tunicamycin (2 μg/ml) (40Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Proteasomal Degradation of CPY*-HA, 6myc-Hmg2, and ΔssCPY*-GFP, Cell Fractionation and Protease Protection Assay—Degradation at 30 or 37 °C was followed by immunoblotting of cells collected at indicated time points after addition of cycloheximide (150 μg/ml), lysed and resolved by SDS-PAGE (9Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell Biol. 2002; 22: 626-634Crossref PubMed Scopus (471) Google Scholar). Dislocation of CPY*-HA was monitored by cell fractionation and protease protection assay, as previously described (12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, postnuclear supernatant (1,000 × g, 10 min) from disrupted spheroplasts was treated with trypsin (0.5 mg/ml, 30 min, 4 °C) with or without Triton X-100 (1% v/v), and microsomal pellets and cytosolic supernatants were separated by centrifugation (10,000 × g, 30 min, 4 °C). CPY*-HA immunoprecipitated with an anti-HA antibody was resolved by SDS-PAGE and immunoblotted. Immunoprecipitation and Immunoblotting—Immunoprecipitation of detergent-dissolved samples and immunoblotting of immunoprecipitated proteins or total cellular proteins were previously described (9Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell Biol. 2002; 22: 626-634Crossref PubMed Scopus (471) Google Scholar, 12Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Primary antibodies: mouse anti-HA (clone 12CA5); mouse anti-Myc (clone 9E10); rabbit anti-Cdc48 (generously provided by K. U. Fröhlich); mouse anti-CPY (clone 10A5-B5; New Biotechnology); chicken anti-Rpt1; rabbit anti-Rpt5 (Affinity Biomol); rabbit anti-Sec61 (generously provided by N. Nelson). Horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (Jackson), goat-anti-rabbit IgG (Sigma); rabbit anti-chicken (Chemicon). The horseradish peroxidase was visualized by enhanced chemiluminescence (ECL) reaction. Proteasome In-gel Peptidase Activity Assay—Non-denaturing PAGE of intact 26S proteasomes directly resolved from whole cell extracts, gels incubation with the fluorogenic peptide Suc-LLVY-AMC as a peptidase substrate, and the in-gel visualization of proteasome assembly species by UV light were previously described (15Bajorek M. Finley D. Glickman M.H. Curr. Biol. 2003; 13: 1140-1144Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar, 41Leggett D.S. Glickman M.H. Finley D. Methods Mol. Biol. 2005; 301: 57-70PubMed Google Scholar). To estimate the total amount of all proteasome species, regardless of their relative activities, salt-induced dissociation of 20S CP and 19S RP was carried out prior to subjecting samples to non-denaturing PAGE, followed by 20S CP activation in gels incubated for 10 min in buffer with Suc-LLVY-AMC substrate and supplemented with 0.02% (w/v) SDS as described previously (15Bajorek M. Finley D. Glickman M.H. Curr. Biol. 2003; 13: 1140-1144Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar, 41Leggett D.S. Glickman M.H. Finley D. Methods Mol. Biol. 2005; 301: 57-70PubMed Google Scholar). Proteasomal Rpt Subunits Are Not Identical and Indeed Equivalent Mutations Are Distinctively Manifested in Turnover of Luminal and Membrane ERAD Substrates—The obligatory ATP-requiring steps in proteasomal degradation are assumed to include substrate unfolding by the 19S and translocation into the 20S (25Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.M. Finley D. Schmidt M. Nat. Cell Biology. 1999; 1: 221-226Crossref PubMed Scopus (388) Google Scholar, 26Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (657) Google Scholar, 28Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 42Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 43Benaroudj N. Zwickl P. Seemuller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). However, the six Rpt AAA-ATPases at the base of the 19S are functionally non-equivalent, as shown by a comparative study of equivalent conservative mutations in the invariant lysine in the Walker A motif of each of the six RPT genes. These strains exhibit diverse phenotypes vis-à-vis growth sensitivity to temperature and amino acid analogs, protein degradation in vivo and proteolytic activities of purified proteasomes in vitro (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). To assess whether their non-equivalence could be extended to ERAD, possibly uncovering unique roles for proteasomal ATPases in this pathway, we tested the degradation of ERAD substrates in wild-type, in rpt2RF, rpt3R, rpt4R, rpt5R, rpt6R (harboring a conservative lysine-to-arginine substitution) and in rpt1S (harboring a non-conservative substitution) (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar). Using the above mutants, we assessed the contribution of each Rpt subunit to ERAD of two well-established topologically distinct ERAD substrates. Luminal CPY*-HA is an HA-tagged version of the unstable point mutant (G255R) of the soluble vacuolar protein carboxypeptidase Y (6Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (616) Google Scholar), while membrane 6myc-Hmg2 is an unstable Myc-tagged version of the 3-hydroxy-3-methylglutaryl-CoA reductase (44Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (465) Google Scholar). As shown in Fig. 1, we estimated the half-life of CPY*-HA in each rpt mutant by employing the cycloheximide chase assay. In the wild-type (SUB62), t½ was under 1 h. Similar degradation rates were measured in rpt1, rpt3, and rpt5 mutants (t½ ∼ 55 min), and only a slightly reduced turnover rate was observed in rpt6 mutant (t½ ∼ 80 min). However, degradation of CPY*-HA was markedly impaired in rpt2RF and rpt4R strains with extended t½ values approaching 6 h (Fig. 1A). This result clearly points to non-equivalence of Rpt subunits also in ERAD. We next determined whether the impaired degradation of CPY*-HA in rpt2RF and rpt4R reflected a general ERAD phenomenon, or was specific to this substrate. For this purpose, we followed ERAD of membrane 6myc-Hmg2. Once again, 6myc-Hmg2 was rapidly degraded in wild-type (t½ ∼ 1.8 h), yet markedly stabilized in rpt2RF and to an even greater extent in rpt4R, with extended t½ values approaching 5 h and 20 h, respectively (Fig. 1B). The other rpt mutations affected 6myc-Hmg2 degradation only slightly, with t½ values ranging from 1.5 to 2.2 h (Fig. 1B). Hence the degradation patterns of membrane 6myc-Hmg2 or luminal CPY*-HA resemble each other, indicating that only Rpt2 and Rpt4 mutants are manifested in impaired ERAD. Turnover of a Cytosolic Substrate Distinguishes Rpt2, a Participant of General Proteasomal Degradation, from Rpt4, Which Contributes Preferentially to ERAD—The stabilization of ERAD substrates in rpt2RF concurs with the central role of Rpt2 in mediating channel opening into the gated proteolytic chamber (28Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar) and as such in promoting degradation of multiple substrates tested so far (31Rubin D.M. Glickman M.H. Larsen C.N. Dhruvakumar S. Finley D. EMBO J. 1998; 17: 4909-4919Crossref PubMed Scopus (265) Google Scholar, 45Sears C. Olesen J. Rubin D. Finley D. Maniatis T. J. Biol. Chem. 1998; 273: 1409-1419Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 46Guttmann-Raviv N. Martin S. Kassir Y. Mol. Cell Biol. 2002; 22: 2047-2056Crossref PubMed Scopus (62) Google Scholar, 47Kaplun L. Ivantsiv Y. Kornitzer D. Raveh D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10077-10082Crossref PubMed Scopus (47) Google Scholar). The stabilization of ERAD substrates in rpt4R may also reflect a general role in proteasomal degradation. Alternatively, Rpt4 may contribute preferentially to ERAD, as implied by the pronounced stabilization of both ERAD substrates we tested. To differentiate between these options, degradation of a cytosolic substrate of the proteasome was monitored. As the closest relative of the luminal ERAD substrate CPY*-HA, we selected ΔssCPY*-GFP (34Medicherla B. Kostova Z. Schaefer A. Wolf D.H. EMBO Rep. 2004; 5: 692-697Crossref PubMed Scopus (170) Google Scholar), as both substrates encompass an identical CPY* domain (the HA and GFP tags exerted no effect on degradation). Lacking a signal sequence, ΔssCPY*-GFP remained in the cytosol and, as expected, it was stabilized in the proteasomal β-subunit temperature sensitive mutant pre1-1 at 37 °C (Fig. 2A), confirming its proteasomal degradation. Clearly, in the rpt2RF mutant ΔssCPY*-GFP was stabilized and the 30-50-min half-life of this cytosolic substrate was extended to values approaching 4 h (Fig. 2B). Interestingly, degradation of cytosolic ΔssCPY*-GFP proceeded unabated" @default.
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• W1994485108 date "2008-03-01" @default.
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• W1994485108 title "A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates" @default.
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