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• W2103756446 abstract "A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function. A critical aspect of E3 ubiquitin ligase function is the selection of a particular E2 ubiquitin-conjugating enzyme to accomplish ubiquitination of a substrate. We examined the requirements for correct E2-E3 specificity in the RING-H2 ubiquitin ligase Hrd1p, an ER-localized protein known to use primarily Ubc7p for its function. Versions of Hrd1p containing the RING motif from homologous E3s were unable to carry out Hrd1p function, revealing a requirement for the specific Hrd1p RING motif in vivo. An in vitro assay revealed that these RING motifs were sufficient to function as ubiquitin ligases, but that they did not display the E2 specificity predicted from in vivo results. We further refined the in vitro assay of Hrd1p function by demanding not only ubiquitin ligase activity, but also specific activity that recapitulated both the E2 specificity and RING selectivity observed in vivo. Doing so revealed that correct E2 engagement by Hrd1p required the presence of portions of the Hrd1p soluble cytoplasmic domain outside the RING motif, the placement of the Hrd1p ubiquitin ligase in the ER membrane, and presentation of Ubc7p in the cytosolic context. We confirmed that these conditions supported the ubiquitination of Hrd1p itself, and the transfer of ubiquitin to the prototype substrate Hmg2p-GFP, validating Hrd1p self-ubiquitination as a viable assay of ligase function. Ubiquitin is a covalent protein tag that alters the stability or behavior of a growing list of proteins (1Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 9-20Crossref PubMed Scopus (1661) Google Scholar, 2Nakayama K.I. Nakayama K. Nat. Rev. Cancer. 2006; 6: 369-381Crossref PubMed Scopus (1145) Google Scholar, 3Huang T.T. D'Andrea A.D. Nat. Rev. Mol. Cell. Biol. 2006; 7: 323-334Crossref PubMed Scopus (219) Google Scholar, 4Staub O. Rotin D. Physiol. Rev. 2006; 86: 669-707Crossref PubMed Scopus (181) Google Scholar). Covalent attachment of ubiquitin to target proteins occurs by a cascade of enzymes, beginning with a ubiquitin-activating enzyme (E1) 3The abbreviations used are: E1, ubiquitin-conjugating enzyme; E2, ubiquitin-activating enzyme; E3, ubiquitin ligase; ERAD, endoplasmic reticulum-associated degradation; CBD, chitin-binding domain; Hmg-CoA, 3-hydroxy-3-methylglutaryl CoA; HA, hemagglutinin; SOEing, strand overlap extension; GFP, green fluorescent protein; AEBSF, 4-(2-aminoethyl)benzenesulphonyl fluoride; PMSF, phenylmethylsulfonyl fluoride; TPCK, 1-chloro-3-tosylamido-4-phenyl-2-butanone; MOPS, 3-(N-morpholino) propanesulfonic acid; DTT, dithiothreitol; WT, wild type; GST, glutathione S-transferase. 3The abbreviations used are: E1, ubiquitin-conjugating enzyme; E2, ubiquitin-activating enzyme; E3, ubiquitin ligase; ERAD, endoplasmic reticulum-associated degradation; CBD, chitin-binding domain; Hmg-CoA, 3-hydroxy-3-methylglutaryl CoA; HA, hemagglutinin; SOEing, strand overlap extension; GFP, green fluorescent protein; AEBSF, 4-(2-aminoethyl)benzenesulphonyl fluoride; PMSF, phenylmethylsulfonyl fluoride; TPCK, 1-chloro-3-tosylamido-4-phenyl-2-butanone; MOPS, 3-(N-morpholino) propanesulfonic acid; DTT, dithiothreitol; WT, wild type; GST, glutathione S-transferase. hydrolyzing ATP to form a thioester-linked ubiquitin-bound intermediate. The E1 next passes its ubiquitin to a ubiquitin-conjugating enzyme (E2), again as a thioester-linked intermediate. Finally, ubiquitination of the target protein is brokered by a ubiquitin ligase (E3) that facilitates transfer of ubiquitin from the E2 to a lysine on the target protein (or a previously added ubiquitin) to form an isopeptide bond. In vivo the ubiquitin ligase activity of a given E3 is not universally supported by all E2s (5Hochstrasser M. Annu Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1452) Google Scholar, 6Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6825) Google Scholar). A typical E3 will function with only one or two of many E2s in vivo (7Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2897) Google Scholar, 8Seol J.H. Feldman R.M. Zachariae W. Shevchenko A. Correll C.C. Lyapina S. Chi Y. Galova M. Claypool J. Sandmeyer S. Nasmyth K. Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (354) Google Scholar, 9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar, 10Swanson R. Locher M. Hochstrasser M. Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (371) Google Scholar, 11Gardner R.G. Nelson Z.W. Gottschling D.E. Cell. 2005; 120: 803-815Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Thus, the compatibility between E3 and E2 is a critical aspect of this enzyme cascade. Many E3s share a zinc-binding sequence called the RING motif (12Fang S. Lorick K.L. Jensen J.P. Weissman A.M. Semin. Cancer Biol. 2003; 13: 5-14Crossref PubMed Scopus (115) Google Scholar). This characteristic sequence, along with several variants (13Hatakeyama S. Yada M. Matsumoto M. Ishida N. Nakayama K.I. J. Biol. Chem. 2001; 276: 33111-33120Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 14Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), is found in a large number of known or putative E3s, where it is required for ubiquitin ligase activity both in vivo and in vitro (8Seol J.H. Feldman R.M. Zachariae W. Shevchenko A. Correll C.C. Lyapina S. Chi Y. Galova M. Claypool J. Sandmeyer S. Nasmyth K. Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (354) Google Scholar, 9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar, 11Gardner R.G. Nelson Z.W. Gottschling D.E. Cell. 2005; 120: 803-815Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 15Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (938) Google Scholar, 16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar, 17Chen B. Mariano J. Tsai Y.C. Chan A.H. Cohen M. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 341-346Crossref PubMed Scopus (172) Google Scholar). Unlike the HECT domain ligases, the RING ligases and their variants do not form a covalent adduct with ubiquitin during catalysis (18Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1256) Google Scholar, 19Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1010) Google Scholar). Despite the prevalence of RING motifs among the growing number of ubiquitin ligases, and their necessity for ligase function in these proteins, the role of the RING motif in promoting E2 specificity and ubiquitin transfer is not fully understood. Structural analyses suggest that residues in the RING motif make contact with E2s, but so too do some residues outside the RING motif (20Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, 21Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Crossref PubMed Scopus (1157) Google Scholar, 22Brzovic P.S. Keeffe J.R. Nishikawa H. Miyamoto K. Fox 3rd, D. Fukuda M. Ohta T. Klevit R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5646-5651Crossref PubMed Scopus (282) Google Scholar, 23Katoh S. Hong C. Tsunoda Y. Murata K. Takai R. Minami E. Yamazaki T. Katoh E. J. Biol. Chem. 2003; 278: 15341-15348Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is not clear whether the RING motif alone is sufficient to specifically engage an E2 and stimulate ubiquitination activity, or whether other in cis determinants outside the RING motif contribute to E2 selectivity. We have addressed these questions for the case of the ubiquitin ligase Hrd1p. A significant component of protein degradation in eukaryotes occurs at the surface of the ER and is generally referred to as ERAD, for ER-associated degradation. ERAD is responsible for degradation of a variety of integral membrane and luminal proteins in the ER (24Brodsky J.L. McCracken A.A. Semin. Cell Dev. Biol. 1999; 10: 507-513Crossref PubMed Scopus (298) Google Scholar). The Hrd1p ubiquitin ligase in Saccharomyces cerevisiae is one of several E3s that mediate ERAD (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar, 10Swanson R. Locher M. Hochstrasser M. Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (371) Google Scholar, 25Haynes C.M. Caldwell S. Cooper A.A. J. Cell Biol. 2002; 158: 91-101Crossref PubMed Scopus (114) Google Scholar), with homologs in all eukaryotes (26Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Crossref PubMed Scopus (355) Google Scholar, 27Amano T. Yamasaki S. Yagishita N. Tsuchimochi K. Shin H. Kawahara K. Aratani S. Fujita H. Zhang L. Ikeda R. Fujii R. Miura N. Komiya S. Nishioka K. Maruyama I. Fukamizu A. Nakajima T. Genes Dev. 2003; 17: 2436-2449Crossref PubMed Scopus (161) Google Scholar). Hrd1p is responsible for the degradation of the yeast HMG-CoA reductase isozyme Hmg2p (28Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (460) Google Scholar), and a variety of misfolded ER proteins (29Bordallo J. Plemper R.K. Finger A. Wolf D.H. Mol. Biol. Cell. 1998; 9: 209-222Crossref PubMed Scopus (297) Google Scholar, 30Plemper R.K. Egner R. Kuchler K. Wolf D.H. J. Biol. Chem. 1998; 273: 32848-32856Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 31Wilhovsky S. Gardner R. Hampton R. Mol. Biol. Cell. 2000; 11: 1697-1708Crossref PubMed Scopus (95) Google Scholar). Hrd1p consists of an N-terminal multi-spanning membrane anchor and a cytoplasmic C-terminal region bearing a RING-H2 motif. The cytoplasmic portion is required for Hrd1p-dependent ERAD in vivo (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar) and functions autonomously as a ubiquitin ligase in vitro (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar). Hrd1p is complexed with the ER integral membrane protein Hrd3p. One function of Hrd3p is to promote Hrd1p stability. In the absence of Hrd3p, Hrd1p undergoes rapid degradation mediated by the Hrd1p RING-H2 motif resulting in Hrd1p levels too low to sustain ERAD (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar). If Hrd1p levels are elevated in the absence of Hrd3p, ERAD will proceed, indicating that Hrd1p is a key protein of HRD pathway ubiquitination (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar). The cytoplasmic C-terminal RING-H2 domain of Hrd1p is exposed to numerous ubiquitin E2s; yet, Hrd1p only employs Ubc7p, and to a much lesser extent Ubc1p, in the execution of its function (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar). This same E2 specificity is observed for Hrd1p self-ubiquitination in the absence of Hrd3p (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar). However, the Hrd1p RING domain will function in vitro with E2s that are not used in vivo (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar), leading us to wonder which features of Hrd1p contribute to its high selectivity for Ubc7p in vivo. In particular, we were interested in developing biochemical approaches for studying Hrd1p action that would faithfully recapitulate this selectivity in biochemically tractable conditions. We have determined that cis-acting portions of Hrd1p, the membrane anchoring protein Cue1p, and the placement of Hrd1p in the ER membrane bilayer are all critical to reconstituting in vitro the function of Ubc7p with Hrd1p. Recombinant DNA—Detailed plasmid information is available in supplemental data. PCR primer information will be provided upon request. All DNA segments synthesized by PCR were verified by sequencing. The production of coding regions was described for Hrd1p-3HA and C399S Hrd1p-3HA (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar), as well as Ubc7p-2HA (32Gardner R.G. Shearer A.G. Hampton R.Y. Mol. Cell. Biol. 2001; 21: 4276-4291Crossref PubMed Scopus (105) Google Scholar). gp78, hsHrd1, and Praja1 RING motifs were amplified by PCR from published template plasmids (15Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (938) Google Scholar, 33Kikkert M. Doolman R. Dai M. Avner R. Hassink G. van Voorden S. Thanedar S. Roitelman J. Chau V. Wiertz E. J. Biol. Chem. 2004; 279: 3525-3534Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), and joined to Hrd1p sequences by a PCR SOEing method to precisely replace the native Hrd1p RING motif (34Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6812) Google Scholar, 35Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2634) Google Scholar), and subcloned into plasmids containing Hrd1p-3HA to yield gp78, hsHrd1, or Praja1 RING chimera in otherwise full-length Hrd1p-3HA. These were then subcloned into yeast expression plasmids with either the native HRD1 promoter or the strong TDH3 promoter. Hrd1p-Δpro was made using PCR SOEing to join the sequences of Hrd1p on either side of the proline-flanked deletion (see Fig. 1), and subcloned into appropriate Hrd1p-3HA plasmids. C399A-Hrd1p was made by PCR SOEing, and was found to be as potent a RING mutant as C399S-Hrd1p (data not shown). All GST fusions were expressed from the pET42b(+) bacterial expression plasmid (Novagen). The isolated RING motifs were amplified by PCR from plasmids above and subcloned into pET42b(+). pRH1466, the plasmid expressing GST-R-C, was previously described (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar). GST-N-R and GST-N-R-C were made by PCR of the appropriate sequence from Hrd1p plasmid and subcloning into pRH1466. GST-N-c399s-C and GST-N-gp78-C were made by PCR of sequence encoding the mutant C399S-Hrd1p or chimeric gp78-RING-Hrd1p. The c399 refers to the last cysteine of the Hrd1p RING that normally occupies position 399 of full-length Hrd1p. The Ubc7p or Ubc7p-2HA coding region was amplified by PCR and subcloned into pTYB2 (New England Biolabs) to produce the Ubc7p-Chitin Binding Domain/Intein fusion vector pRH1946. ΔtmCue1p, which lacks amino acids 2–22 of Cue1p (and thus the included transmembrane span) was amplified by PCR from pTX129 (36Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (328) Google Scholar) and cloned into a pET bacterial expression vector. Then, the ribosomal binding site and ΔtmCue1p were amplified by PCR and cloned behind Ubc7p-CBD/Intein in pRH1946 to produce pRH2061, with a polycistronic message encoding both Ubc7p-CBD/Intein and ΔtmCue1p proteins in one inducible operon. His6-tagged mouse UBA1 (E1) and HUBC4 were purified from bacterial lysates as described previously (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar, 37Mori S. Tanaka K. Kanaki H. Nakao M. Anan T. Yokote K. Tamura K. Saito Y. Eur. J. Biochem. 1997; 247: 1190-1196Crossref PubMed Scopus (13) Google Scholar, 38Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (911) Google Scholar). Strains and Media—Yeast were cultured at 30 °C as described (28Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (460) Google Scholar, 39Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (181) Google Scholar), in minimal media with 2% glucose and amino acid supplements. Detailed strain information is presented in supplemental data. All yeast strains were derived from the same genetic background used in our previous work (28Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (460) Google Scholar, 39Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (181) Google Scholar). Strains for evaluating the in vivo degradation of Hmg2p-GFP were derived from the previously described RHY853 (16Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar), expressing Hmg2-GFP and the independently expressed catalytic domain of Hmg2p as its sole source of HMG-CoA reductase. HRD1 was replaced in RHY853 with the G418-resistance marker kanMX (40Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1346) Google Scholar) to produce RHY2814. The various HA epitope-tagged Hrd1p chimeric RING plasmids or controls were integrated into this hrd1Δ strain at the TRP1 locus. To evaluate Hrd1p degradation, HRD3 was deleted in RHY2814 with the selectable LEU2 marker to produce RHY3005. Into this hrd1Δhrd3Δ strain, the various Hrd1p RING replaced plasmids or controls were also integrated at the TRP1 locus. To evaluate Ubc7p dependence of Hrd1p degradation, UBC7 was deleted in RHY3005 with the nourseothricin-(ClonNat) resistance marker natMX (41Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1370) Google Scholar) to produce RHY3559, into which Hrd1p RING replaced plasmids or controls were integrated at the TRP1 locus. Strains used to produce microsomal membranes for the in vitro assay were pep4Δ ubc7Δ hrd1Δ, and expressed Hmg2p-GFP. The full-length Hrd1p-3HA chimeras tested in microsomes were expressed in these strains from the strong TDH3 promoter by integration of the appropriate plasmid at the TRP1 locus. cue1Δ nulls were generated from these strains by deletion of CUE1 with the nourseothricin (ClonNat) resistance marker natMX (41Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1370) Google Scholar). Strains for the production of cytosol were also pep4Δ hrd1Δ ubc7Δ and included either empty vector or Ubc7p-2HA expressed from the TDH3 promoter. Flow Cytometry—Log phase cultures (A600 <0.5) grown in minimal medium at 30 °C were transferred to flow cytometer sample tubes and measured with a Becton Dickinson FACScalibur instrument. Flow microfluorimetric data were analyzed and histograms were generated using CellQuest flow cytometry software. In all cases, histograms represented 20,000 individual cells. Cycloheximide Chase Degradation Assay—This assay was performed as described (39Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (181) Google Scholar). Briefly, log phase cultures of cells expressing HA epitope-tagged Hrd1p or RING variants were treated with 50 μg/ml cycloheximide to arrest protein synthesis. At indicated times, 1 OD of log phase cells were harvested, lysed, and 0.1 OD equivalents were resolved by SDS-PAGE and immunoblotted for epitope-tagged protein. Protein Purification—All recombinant proteins were expressed in Rosetta(DE3) Escherichia coli (Novagen) grown in LB with appropriate antibiotics. 0.6 OD/ml cultures were induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 12–16 h at 15 °C. Bacterial pellets were harvested, washed in normal saline (0.9 m NaCl), and frozen at –80 °C. Pellets were thawed and resuspended in extraction buffers as described below, and lysed using a Branson Sonifier 450 (VWR) with six rounds of 30 s sonication/30 s ice incubation. After affinity column purification, elution, and concentration, proteins were dialyzed into buffer HDB containing 10% glycerol. Single use aliquots were flash-frozen with liquid nitrogen and stored at –80 °C. Recombinant protein concentrations were determined by Coomassie staining of SDS-PAGE resolved samples and comparison to bovine serum albumin. His Tag Purification of E1 and HUBC4—Bacterial pellets from 2-liter cultures expressing His6 mouse UBA1 or HUBC4 were resuspended in 40 ml of His-Extraction Buffer (50 mm sodium phosphate pH 7.4, 300 mm NaCl, 0.5% Nonidet P-40, 5% glycerol) with protease inhibitors (13 μm AEBSF, 3.6 μm TPCK, 2.6 μm leupeptin, 1.8 μm pepstatin, 0.56 mm 6-aminohexanoic acid, 0.56 mm benzamidine, and 2.5 mm 2-mercaptoethanol), sonicated as above, and centrifuged at 12,000 × g for 20 min in an SS34 rotor. The supernatant was transferred to a new tube with 0.5 ml of Talon Cell-Thru resin (BD Biosciences) equilibrated in His-Extraction Buffer, and gently nutated for 20 min at room temperature. The resin was centrifuged (3000 × g) and washed with 10 ml of His-Extraction Buffer and protease inhibitors above for 10 min at room temperature two times. The washed resin was then transferred to a 1-cm diameter column and washed with 30 ml of His-Wash Buffer (50 mm sodium phosphate, pH 7.8, 300 mm NaCl, 5 mm imidazole, 5% glycerol, 10 mm 2-mercaptoethanol). His-tagged E1 was eluted from the resin with 3 ml of His-Wash Buffer + 150 mm imidazole, and was collected in 500-μl fractions and analyzed by Bradford assay with bovine serum albumin standard. Fractions with more than 0.1 mg/ml protein were pooled and concentrated with Amicon Ultra-15 5,000 MWCO filters (Millipore). Concentrated protein was dialyzed over 24 h with 3 × 1 liter HDBG (25 mm HEPES, 0.7 mm sodium phosphate, 137 mm NaCl, 5 mm KCl, pH 7.4, 10% glycerol) in a 3-ml 10,000 MWCO Slide-A-Lyser cassette (Pierce). GST Protein Purification—Each bacterial pellet from 1 liter of culture expressing a GST protein was resuspended in 25 ml of buffer HDB (25 mm HEPES, 0.7 mm sodium phosphate, 137 mm NaCl, 5 mm KCl, pH 7.4) + protease inhibitors (7.5 mm EDTA, 1.5 mm PMSF, 10 μm leupeptin, 7 μm pepstatin, 28 μm TPCK, 130 μm AEBSF, 2.5 mm 6-aminohexanoic acid, 2.5 mm benzamidine, and 7.5 mm DTT), and sonicated as above. To this was added 6 ml of 2.5 m NaCl, 600 μl1 m sodium phosphate, pH 7.4, and 1.2 ml of 25% Triton X-100. This was nutated 20 min at 4 °C, and centrifuged for 1 h at 40,000 × g in SS34 rotor. Buffer HDBW (HDB + 20 mm sodium phosphate, pH 7.4, 10 mm 2-mercaptoethanol, 1% Triton X-100.) was added to the supernatant to a final volume of 50 ml along with 1 ml of glutathione-Sepharose-4B resin (Amersham Biosciences) and nutated for 1 h at 4 °C. The resin was transferred to a 1-cm diameter column and washed with 10 ml of each of the following buffers: HDBW with 1.25 mm PMSF and 5 mm EDTA; HDBW with 1.25 mm PMSF, 5 mm EDTA, and 0.5 m NaCl; HDBW with 0.5% deoxycholate; HDBW with 0.5 m NaCl; and Final Wash (40 mm Tris, pH 8.0, 50 mm NaCl, 10 mm 2-mercaptoethanol). Protein was eluted in 1-ml fractions of Elution Buffer (20 mm Tris, pH 8, 10 mm 2-mercaptoethanol, 20 mm reduced glutathione) incubated with column resin for 10 min before recovery. Fractions were analyzed by Bradford assay with bovine serum albumin standard. Fractions with more than 0.1 mg/ml protein were pooled and concentrated with Amicon Ultra-15 5,000 MWCO filters (Millipore). Concentrated protein was dialyzed for 24 h with 3 × 1 liter HDBG (25 mm HEPES, 0.7 mm sodium phosphate, 137 mm NaCl, 5 mm KCl, pH 7.4, 10% glycerol) in a 0.5 ml 3,000 MWCO Slide-A-Lyser cassette (Pierce). Intein/Chitin Binding Domain Fusion Purification—Each bacterial pellet from 1 liter of culture expressing an Intein/CBD fusion was resuspended in 25 ml of Intein Lysis Buffer (ILB: 50 mm Tris, pH 8.0, 500 mm NaCl, 1 mm EDTA, 0.1% Triton X-100) with protease inhibitors (260 μm AEBSF, 105 μm leupeptin, 73 μm pepstatin, 142 μm TPCK), and sonicated as above. Lysate was centrifuged at 20,000 × g for 30 min in an SS34 rotor. Supernatant was filtered through 0.45-μm and 0.2-μm filters, and added to 15 ml of chitin beads (New England Biolabs) equilibrated in ILB, and nutated for 90 min at 4 °C. The adsorbed resin was placed in a 2.5-cm column and washed with 350–400 mls of ILB. Next, the resin was nutated in 10 ml of ILB + 50 mm DTT for 20 h at 4 °C to promote intein cleavage, and chitin beads were washed with ILB to collect intein-cleaved proteins. 40 ml of fluid were collected and concentrated using Amicon Ultra-15 5,000 MWCO filters (Millipore). Concentrated protein was dialyzed against 3 × 1 liter HDBG (25 mm HEPES, 0.7 mm sodium phosphate, 137 mm NaCl, 5 mm KCl, pH 7.4, 10% glycerol) for 24 h in a 0.5 ml 3,000 MWCO Slide-a-Lyser cassette (Pierce). Proteins were ultracentrifuged at 100,000 × g to remove any aggregates, and supernatant was aliquoted as above. In Vitro Ubiquitination—Ubiquitin was resuspended from lyophilized powder in Ubiquitin Storage Buffer (50 mm Tris, pH 7.5, 50 mm NaCl, 10% glycerol) and frozen. Reactions were performed in 1× ubiquitination buffer (50 mm Tris, pH 7.5, 2.5 mm MgCl2, 0.5 mm DTT) with 3 mm ATP, 80 μg/ml ubiquitin, 6 μg/ml E1, 20 μg/ml E2, in a total volume of 15 μl. Reactions mixtures were prepared on ice, then incubated at 30 °C for 2 h, and stopped with an equal volume of 2× sample buffer (4% SDS (w/v), 8 m urea, 75 mm MOPS, pH 6.8, 200 mm DTT, 0.2 mg/ml bromphenol blue) and analyzed by SDS-PAGE and anti-ubiquitin immunoblotting. Microsome Preparation and Ubiquitination—20 A600 units of log phase cells grown in minimal media were harvested and resuspended in 400 μl of ice-cold Membrane Fractionation Buffer (MFB: 20 mm Tris, pH 7.5, 0.1 m NaCl, 0.3 m sorbitol) with protease inhibitors (260 μm AEBSF, 105 μm leupeptin, 73 μm pepstatin, 142 μm TPCK). Glass beads were added to just below the liquid level. Lysis was performed at 4 °C with six cycles of 1 m in vortexing (max speed) and 1 min incubation on ice. Lysate was harvested by removing supernatant from beads, and washing beads twice with 400 μl of MFB, pooling the washes and lysate. The resulting pooled lysate was cleared by repeated 10-s microcentrifuge pulses to remove unlysed cells and large debris. The cleared supernatant contains microsome membranes, which were harvested by centrifugation at 21,000 × g for 30 min. Microsome pellets were resuspended in 60 μl of Ubiquitination Buffer, and the yield from 5 OD of cells (15 μl) was added to each reaction. Reactions were performed in Ubiquitination Buffer with 6 μg/ml E1, 40 μg/ml E2 (except as noted in E2 dilution experiments), 160 μg/ml ubiquitin, and 3 mm ATP in 60-μl reactions. Reaction mixes were prepared on ice, then incubated at 30 °C for 2 h. Reactions were stopped with 200 ml of SUME (1%w/v SDS, 8 m urea, 10 mm MOPS, pH 6.8, 10 mm EDTA) with protease inhibitors above and 5 mm N-ethylmaleimide, followed by addition of 600 μl of IP buffer (15 mm sodium phosphate, 150 mm NaCl, 10 mm EDTA, 2% Triton X-100, 0.1% SDS, 0.5% deoxycholate), immunoprecipitation of Hrd1p as described (9Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (379) Google Scholar), and immunoblotting the SDS-PAGE resolved immunoprecipitate for ubiquitin with anti-ubiquitin antibodies (Zymed Laboratories, South San Francisco, CA) or for Hrd1p with anti-HA ascites fluid (Jackson ImmunoResearch). Microsome Ubiquitination Assay with Cytosol—Cytosol and microsomes were prepared as previously described (42Flury I. Garza R. Shearer A. Rosen J. Cronin S. Hampton R.Y. EMBO J" @default.
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• W2103756446 date "2006-12-01" @default.
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• W2103756446 title "Determinants of RING-E2 Fidelity for Hrd1p, a Membrane-anchored Ubiquitin Ligase" @default.
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• W2103756446 doi "https://doi.org/10.1074/jbc.m608174200" @default.
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