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Cue1p Is an Activator of Ubc7p E2 Activity in Vitro and in Vivo

2008; Elsevier BV; Volume: 283; Issue: 19 Linguagem: Inglês

10.1074/jbc.m801122200

ISSN

1083-351X

Autores

Omar A. Bazirgan, Randolph Y. Hampton,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

Ubc7p is a ubiquitin-conjugating enzyme (E2) that functions with endoplasmic reticulum (ER)-resident ubiquitin ligases (E3s) to promote endoplasmic reticulum-associated degradation (ERAD). Ubc7p only functions in ERAD if bound to the ER surface by Cue1p, a membrane-anchored ER protein. The role of Cue1p was thought to involve passive concentration of Ubc7p at the surface of the ER. However, our biochemical studies of Ubc7p suggested that Cue1p may, in addition, stimulate Ubc7p E2 activity. We have tested this idea and found it to be true both in vitro and in vivo. Ubc7p bound to the soluble domain of Cue1p showed strongly enhanced in vitro ubiquitination activity, both in the presence and absence of E3. Cue1p also enhanced Ubc7p function in vivo, and this activation was separable from the established ER-anchoring role of Cue1p. Finally, we tested in vivo activation of Ubc7p by Cue1p in an assay independent of the ER membrane and ERAD. A chimeric E2 linking Ubc7p to the Cdc34p/Ubc3p localization domain complemented the cdc34-2 TS phenotype, and co-expression of the soluble Cue1p domain enhanced complementation by this chimeric Ubc7p E2. These studies reveal a previously unobserved stimulation of Ubc7p E2 activity by Cue1p that is critical for full ERAD and that functions independently of the well known Cue1p anchoring function. Moreover, it suggests a previously unappreciated mode for regulation of E2s by Cue1p-like interacting partners. Ubc7p is a ubiquitin-conjugating enzyme (E2) that functions with endoplasmic reticulum (ER)-resident ubiquitin ligases (E3s) to promote endoplasmic reticulum-associated degradation (ERAD). Ubc7p only functions in ERAD if bound to the ER surface by Cue1p, a membrane-anchored ER protein. The role of Cue1p was thought to involve passive concentration of Ubc7p at the surface of the ER. However, our biochemical studies of Ubc7p suggested that Cue1p may, in addition, stimulate Ubc7p E2 activity. We have tested this idea and found it to be true both in vitro and in vivo. Ubc7p bound to the soluble domain of Cue1p showed strongly enhanced in vitro ubiquitination activity, both in the presence and absence of E3. Cue1p also enhanced Ubc7p function in vivo, and this activation was separable from the established ER-anchoring role of Cue1p. Finally, we tested in vivo activation of Ubc7p by Cue1p in an assay independent of the ER membrane and ERAD. A chimeric E2 linking Ubc7p to the Cdc34p/Ubc3p localization domain complemented the cdc34-2 TS phenotype, and co-expression of the soluble Cue1p domain enhanced complementation by this chimeric Ubc7p E2. These studies reveal a previously unobserved stimulation of Ubc7p E2 activity by Cue1p that is critical for full ERAD and that functions independently of the well known Cue1p anchoring function. Moreover, it suggests a previously unappreciated mode for regulation of E2s by Cue1p-like interacting partners. A significant component of protein degradation in eukaryotes occurs at the surface of the ER 3The abbreviations used are: ER, endoplasmic reticulum; E1, ubiquitin-conjugating enzyme; E2, ubiquitin-activating enzyme; E3, ubiquitin ligase; ERAD, endoplasmic reticulum-associated degradation; CBD, chitin-binding domain; HA, hemagglutinin; SOEing, strand overlap extension; GFP, green fluorescent protein; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; TPCK, 1-chloro-3-tosylamido-4-phenyl-2-butanone; MOPS, 3-(N-morpholino)propanesulfonic acid; DTT, dithiothreitol; GST, glutathione S-transferase; L-Ubc7p, N-terminally modified linker-Ubc7p-2HA protein; ILB, intein lysis buffer; TS, temperature-sensitive. (1Sommer T. Wolf D.H. FASEB J. 1997; 11: 1227-1233Crossref PubMed Scopus (220) Google Scholar, 2Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (404) Google Scholar, 3Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (364) Google Scholar, 4Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). In this process of ER-associated degradation (ERAD), integral membrane and luminal ER proteins destined for degradation are targeted to the proteasome by the covalent addition of ubiquitin. Attachment of ubiquitin to target proteins occurs by a cascade of enzymes, beginning with a ubiquitin-activating enzyme (E1) hydrolyzing ATP to form a thioester-linked ubiquitin-E1 adduct. The E1 next passes its ubiquitin to a ubiquitin-conjugating enzyme (E2), also as a thioester-linked intermediate. Finally, ubiquitination of the target protein is promoted 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), thus promoting the polyubiquitination of proteins targeted for degradation. In the baker's yeast Saccharomyces cerevisiae, Ubc7p is an E2 required for ERAD mediated by two ER-localized E3s, Hrd1p and Doa10p (5Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar, 6Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar, 7Swanson R. Locher M. Hochstrasser M. Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (377) Google Scholar). Ubc7p is able to engage these ER-localized E3s because Ubc7p is anchored to the ER through interaction with the integral ER membrane protein Cue1p. In fact, Cue1p is required for the Ubc7p-dependent ubiquitination and degradation of substrates in the ER (8Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar, 9Gardner R.G. Shearer A.G. Hampton R.Y. Mol. Cell Biol. 2001; 21: 4276-4291Crossref PubMed Scopus (105) Google Scholar). Because proximity and interaction between E2 and E3 are critical for ubiquitination function (10VanDemark A.P. Hill C.P. Curr. Opin. Struct. Biol. 2002; 12: 822-830Crossref PubMed Scopus (54) Google Scholar), it has been suggested that the main function of Cue1p is to concentrate Ubc7p at the ER membrane surface, thus allowing fruitful engagement of Ubc7p with ER-localized E3s (8Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar). Although genetic experiments have established a requirement for Cue1p in ERAD and biochemical studies have confirmed that Cue1p and Ubc7p interact, the effects of Cue1p on the enzymatic activity of Ubc7p have not been explored. In our previous study of Hrd1p specificity with Ubc7p, the presence of Cue1p enhanced Ubc7p activity in biochemical assays of Ubc7p function, suggesting that Ubc7p may be activated by Cue1p (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Here we directly test the idea that Cue1p stimulates E2 activity of Ubc7p in vitro and in vivo. We examined the nature of the polyubiquitin chains formed in these E3-independent in vitro reactions. We discovered that in vitro, Ubc7p produced lysine 48-linked polyubiquitin chains, and a soluble portion of Cue1p strongly stimulated this Ubc7p activity in the presence or absence of E3. We then designed chimeric proteins to express in vivo that would separate the established anchoring function of Cue1p from its putative activation function and found that both anchoring and Cue1p-based activation were important for Hrd1p-dependent ERAD. We also developed means to assay Ubc7p activity in a context independent of ERAD or the ER membrane and found that Cue1p activated Ubc7p in a manner entirely independent of ER anchoring. Taken together, these results reveal a previously unknown role for Cue1p as an activator of Ubc7p E2 activity and suggest that other E2s may have similar stimulating cofactors. Recombinant DNA—All DNA segments synthesized by PCR were verified by sequencing. The Ubc7p-chitin-binding domain/intein fusion vector pRH1946 was previously described (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Ubc7p-2HA coding region was amplified by PCR and subcloned into pTYB2 (New England Biolabs) to produce the Ubc7p-2HA-chitin-binding domain/intein fusion vector pRH1947. Cue1pΔ™, which lacks amino acids 2–22 of Cue1p (and thus the included transmembrane span) was amplified by PCR from pTX129 (8Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar) and cloned into a pET bacterial expression vector. Then the ribosomal binding site and Cue1pΔ™ were amplified by PCR and subcloned into both pRH1946 and pRH1947 behind the Ubc7p-CBD/intein to produce pRH2061 and pRH2064, whose polycistronic message encoded both Cue1pΔ™ and either untagged or HA-tagged Ubc7p-CBD/intein in one inducible operon. GST was expressed from the pET42b(+) bacterial expression plasmid (Novagen). GST-E3 was the previously described fusion to Hrd1p expressed from pRH1726 (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). His6-tagged mouse UBA1 (E1) and HUBC4 were purified from bacterial lysates as described previously (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 12Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (381) Google Scholar, 13Joazeiro C.A. Wing S.S. Huang H. Leverson J.D. Hunter T. Liu Y.C. Science. 1999; 286: 309-312Crossref PubMed Scopus (916) Google Scholar, 14Mori 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). Ubc7p with two HA epitope tags was expressed in yeast from the strong TDH3 promoter using the previously described vector pRH373 (9Gardner R.G. Shearer A.G. Hampton R.Y. Mol. Cell Biol. 2001; 21: 4276-4291Crossref PubMed Scopus (105) Google Scholar). To express Ubc7p-2HA from the native UBC7 promoter, the identical coding sequence for Ubc7p-2HA was amplified by PCR and subcloned into a yeast expression vector containing the native UBC7 promoter (pRH2193). For expression of Cue1p in yeast, sequence encoding full-length Cue1p was amplified by PCR and subcloned between the TDH3 promoter and three HA epitope tags of an existing yeast expression vector (pRH1334). Membrane-anchored versions of Ubc7p were made by a PCR SOEing method (15Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 16Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). Sequences encoding the N-terminal 22-amino acid transmembrane span of Cue1p and the entire coding region of Ubc7p-2HA were amplified by PCR and joined by PCR SOEing, and this chimeric PCR product was subcloned into a vector allowing expression of membrane-anchored Ubc7p without linker from the strong TDH3 promoter (pRH2190). TM-Ubc7p included amino acids 531–618 of Hmg2p, a portion of the cytosolic linker between the transmembrane domain and conserved cytosolic catalytic domain of Hmg2p. Sequence encoding this 88-amino acid linker was amplified from pRH469 by PCR and joined to sequences encoding the Cue1p transmembrane span and Ubc7p-2HA by PCR SOEing to produce the TM-Ubc7p sequence. This chimeric PCR product was subcloned into a vector, allowing expression of TM-Ubc7p from the strong TDH3 promoter (pRH2191). Similarly, sequence for the linker described above joined to Ubc7p-2HA, without the transmembrane span of Cue1p, was amplified by PCR and subcloned into pRH2191 to produce pRH2457, expressing the N-terminally modified linker-Ubc7p-2HA protein (L-Ubc7p) from the TDH3 promoter. To express Cue1pΔ™ in yeast, the sequence encoding amino acids 23–203 and the adjacent three HA epitope tags was amplified by PCR from pRH1334 and subcloned behind the strong TDH3 promoter in a yeast expression vector (pRH2198). Sequence encoding Cdc34p was amplified from genomic DNA and subcloned into the previously described p416-GPD vector (17Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google Scholar) between the TDH3 promoter and CYC1 terminator to produce pRH1939. The native CDC34 promoter was amplified from pRG721 (Richard G. Gardner, University of Washington) and subcloned into pRH1939 to make pRH1971, expressing Cdc34p from the CDC34 promoter. To make Ubc7p-Cdc34, UBC7 sequence was linked by PCR SOEing to sequence encoding amino acids 171–295 of Cdc34p. The resulting DNA was subcloned into pRH1939 to express Ubc7p-Cdc34 from the TDH3 promoter (pRH1968). pRH1969, expressing the function-blocking C89S mutant version of Ubc7p-Cdc34, was made as above, except Ubc7p sequence was amplified from a template with the C89S point mutation. Ubc7p-encoding sequence was also cloned into pRH1939 to express Ubc7p from the same vector as the other E2s. These E2 constructs were then each subcloned into pRH1971 described above to express them from the native CDC34 promoter; pRH1983 expressed Ubc7p-Cdc34, pRH1985 expressed Ubc7p-Cdc34 with C89S, and pRH1987 expressed Ubc7p. Protein molecular weight prediction was performed using the Compute pI/Mw tool on the ExPASy proteomics server (18Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3550) Google Scholar). Strains and Media—Yeast strains were cultured as described (19Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (466) Google Scholar, 20Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (184) Google Scholar), in minimal media with 2% glucose and amino acid supplements, at 30 °C unless otherwise indicated. Only in the cdc34-2 complementation experiments, strains were grown in synthetic complete media lacking uracil and leucine to maintain plasmid selection. All yeast strains were derived from the same genetic background used in our previous work (19Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (466) Google Scholar, 20Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (184) Google Scholar). Strains for evaluating the in vivo degradation of Hmg2p-GFP were derived from RHY853 (21Gardner 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 (249) Google Scholar), expressing the catalytic domain of Hmg2p as its sole source of 3-hydroxy-3-methylglutaryl-CoA reductase, and Hmg2p-GFP. To test complementation of Ubc7p and Ubc7p-containing constructs, UBC7 was replaced with the selectable HIS3 marker, producing the ubc7Δ strain RHY1848. Constructs expressing Ubc7p, Cue1p, and membrane-anchored versions of Ubc7p were introduced into this strain to test their restoration of Ubc7p function. To examine restoration of both Ubc7p and Cue1p function, the CUE1 gene was replaced in RHY1848 with the nourseothricin (Clon-Nat) resistance marker natMX (22Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1389) Google Scholar) to produce the ubc7Δ cue1Δ strain RHY5917. In this strain, Cue1pΔ™ and TM-Ubc7p were expressed (individually and together) to test restoration of ERAD function. The cdc34-2 strains were generated in the following manner. To convert the native CDC34 locus to the cdc34-2 allele encoding the G58R mutation, the pRG721 plasmid encoding cdc34-2 was integrated into the strain RHY2863 (ade2–101 met2 lys2–801 ura3–52 trp1:hisG leu2Δ his3Δ200) at the CDC34 locus, placing the selectable URA3 gene between two copies of CDC34, one wild-type and one mutant. These cells were grown in the presence of uracil to allow spontaneous recombination between the two CDC34 loci and loss of URA3, leaving only one copy of CDC34. Such strains were selected on media with 5-fluoroorotic acid for absence of URA3 and then screened for temperature sensitivity to identify strains that retained only the mutant cdc34-2 allele. This cdc34-2 strain derived from RHY2863 is RHY3802. RHY3802 was transformed with plasmids to express from the TDH3 promoter Cdc34p, Ubc7p-Cdc34, or Ubc7p, to test complementation of the cdc34-2 TS growth phenotype. RHY3802 was also transformed with plasmids to express from the CDC34 promoter Cdc34p, Ubc7p-Cdc34, or Ubc7p, to test complementation of the cdc34-2 TS growth phenotype. In turn, the CDC34 promoter strains were transformed with empty vector or Cue1pΔ™ expression plasmid to assess the activation of Ubc7p by Cue1p. CUE1 was disrupted in RHY3802 by introducing a knock-out cassette containing the natMX gene, conferring resistance to nourseothricin. Loss of the CUE1 gene was confirmed by PCR, yielding the cdc34-2 cue1Δ strain RHY7371. This strain was then transformed with plasmids to express from the CDC34 promoter Cdc34p or Ubc7p-Cdc34 as above. Cue1pΔ™ expression plasmid was also transformed as above to assess activation of Ubc7p-Cdc34 by Cue1p in a cue1Δ strain. Protein Purification—All recombinant proteins were expressed in Rosetta(DE3) Escherichia coli (Novagen) grown in LB with appropriate antibiotics. E1, all E2s, and E3 were each purified using the appropriate affinity matrix and previously described methods (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Single use aliquots of each protein preparation were flash-frozen with liquid nitrogen and stored at –80 °C for later use. E1 and HUBC4 were expressed with the His6 tag and purified using Talon Cell-Thru resin (BD Biosciences). GST and GST-Hrd1p fusion (E3) were purified using glutathione-Sepharose-4B resin (Amersham Biosciences). To purify Ubc7p alone or to co-purify Ubc7p with co-expressed Cue1pΔ™, Ubc7p was expressed as a chitin-binding domain/intein fusion. 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 before. Lysate was centrifuged at 20,000 × g for 30 min in an SS34 rotor. Supernatant was filtered through 0.45- 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 ml of ILB. Next, the resin was nutated in 10 ml of ILB plus 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 molecular weight cut-off filters (Millipore). Concentrated protein was dialyzed against 3 × 1 liter of 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 molecular weight cut-off Slide-a-Lyzer cassette (Pierce). Proteins were ultracentrifuged at 100,000 × g to remove any aggregates, and supernatant was aliquoted and frozen. For gel filtration analysis, Ubc7p-2HA·Cue1pΔ™ was prepared as above (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) with the following modifications. The protein eluate was collected and concentrated as described and then dialyzed against 3× 1 liter of UbR150 buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 2.5 mm MgCl2, 0.5 mm DTT) for 24 h in a 0.5-ml 3,000 molecular weight cut-off Slide-a-Lyzer cassette (Pierce). This buffer was like ubiquitin reaction buffer with the addition of 150 mm NaCl to reduce nonspecific interactions with the gel filtration resin. The dialyzed protein preparation was centrifuged at 6,000 rpm in an SS34 rotor to precipitate any aggregates prior to gel filtration. 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. Mutant varieties of ubiquitin were purchased from Boston Biochem, Inc. (Cambridge, MA). 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. Protein concentrations were determined by Coomassie staining and comparison with bovine serum albumin standards. In each experiment, proteins common to multiple reactions were added to a reaction mixture and mixed to ensure equal addition of the common components in each reaction. Such partially assembled reactions were aliquoted to individual tubes for the addition of unique reaction components. Reaction mixtures were prepared on ice and 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 immunoblotting or Coomassie staining as indicated. Immunoprecipitation of Ubc7p with Thioester-linked Ubiquitin—Ubiquitin reactions were prepared as above in 50-μl reactions and then incubated at 30 °C for 2 h. Reactions were stopped by adding 100 μl of SUME (1% (w/v) SDS, 8 m urea, 10 mm MOPS, pH 6.8, 10 mm EDTA) with protease inhibitors (260 μm AEBSF, 105 μm leupeptin, 73 μm pepstatin, 142 μm TPCK) and 5 mm N-ethylmaleimide, followed by the 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) with protease inhibitors above. HA epitope antibody-conjugated resin (Covance) was diluted 6-fold in IP buffer with 0.5 mg/ml bovine serum albumin and incubated for 20 min to block the resin. 120 μl of resin/bovine serum albumin slurry was added to each stopped reaction and incubated at 4 °C for 6 h to precipitate HA-tagged Ubc7p. Beads were washed once with IP buffer and twice with IP wash (50 mm NaCl, 10 mm Tris, pH 7.5), aspirated to dryness, and heated in nonreducing sample buffer (4% SDS (w/v), 8 m urea, 75 mm MOPS, pH 6.8, 0.2 mg/ml bromphenol blue) or reducing sample buffer (same as above plus 200 mm DTT). The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for HA epitope or ubiquitin. Assay of E2 Charging by E1—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, and the indicated E2 concentration, in a total volume of 15 μl. Reaction mixtures were prepared on ice, incubated at room temperature for 5 min, and then stopped with an equal volume of non-reducing 2x sample buffer (4% SDS (w/v), 8 m urea, 75 mm MOPS, pH 6.8, 0.2 mg/ml bromphenol blue) and analyzed by SDS-PAGE and immunoblotting for ubiquitin or HA-epitope. Pixel quantitation was performed with Adobe Photoshop version 7.0.1. Protease Protection Assay—Samples of Ubc7p-2HA or Ubc7p-2HA-Cue1pΔ™ were added to 1× ubiquitination buffer at 40 μg/ml on ice, and lyophilized trypsin resuspended in 1× ubiquitination buffer was added at 10 μg/ml. Trypsin digests were then incubated at room temperature, and aliquots were removed at each indicated time, treated with an equal volume of 2× sample buffer with 260 μm AEBSF, and heated to stop proteolysis. Proteolyzed proteins were resolved by SDS-PAGE and immunoblotted for HA epitope. Gel Filtration—Using an AKTA FPLC system, a 102.5-ml, 51 × 1.6-cm Superose 6 gel filtration column was equilibrated with UbR150 buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 2.5 mm MgCl2, 0.5 mm DTT) and 1 mm AEBSF. 0.6 mg of Ubc7p-2HA·Cue1pΔ™ in 1 ml of UbR150 buffer was loaded onto the column, and 1-ml fractions were collected at a flow rate of 0.4 ml/min UbR150 buffer with AEBSF. A portion of each elution fraction with UV absorbance at 280 nm was resolved by SDS-PAGE and Coomassie-stained or immunoblotted as indicated. The gel filtration column was run in identical conditions with a mixture of protein standards to correlate the fraction distribution with molecular weight. Lyophilized RNase A (13.7 kDa), chymotrypsin (25 kDa), ovalbumin (43 kDa), and albumin (67 kDa) protein standards were reconstituted in UbR150 at 2 mg/ml each, and 1 ml of the protein standard mixture was applied to the column and run as before. A portion of each elution fraction with UV absorbance at 280 nm was resolved by SDS-PAGE and Coomassie-stained. 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 BD Biosciences FACScalibur instrument. Flow microfluorimetric data were analyzed, and histograms were generated using CellQuest flow cytometry software. In all cases, histograms represented 10,000 individual cells. Cycloheximide Chase Assay—Log phase cultures (A600 < 0.1) grown in minimal medium at 30 °C were split into three tubes. One was treated with no drug. The other two were exposed to 50 μg/ml cycloheximide for either 30 min or 2 h. Then each sample was transferred to flow cytometer sample tubes and measured as above. Microsome Preparation—Five optical density units of log phase cells grown in minimal media were harvested and resuspended in 200 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 min of vortexing (maximum speed) and 1 min of incubation on ice. Lysate was harvested by removing supernatant from beads and washing beads twice with 200 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. The pellet was resuspended in 100 μl of SUME (1% (w/v) SDS, 8 m urea, 10 mm MOPS, pH 6.8, 10 mm EDTA) with protease inhibitors above. After the addition of 100 μl 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 heating at 65 °C, the samples were analyzed by SDS-PAGE and anti-HA immunoblotting. Whole Cell Lysates—Five optical density units of log phase cells grown in minimal medium were harvested and resuspended in 100 μl of SUME (1% (w/v) SDS, 8 m urea, 10 mm MOPS, pH 6.8, 10 mm EDTA) with protease inhibitors above and vortexed with glass beads for 3 min. Then 100 μl 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) was added, and samples were heated at 65 °C for 10 min and analyzed by SDS-PAGE and immunoblotting. Growth Assay for cdc34-2 Temperature-sensitive Phenotype—Log phase cultures (A600 < 0.5) for each strain tested were grown in synthetic complete liquid medial lacking leucine and uracil at 30 °C. These were normalized to equal A600 and then serially diluted 5-fold and deposited with a 48-pin replicator onto plates of synthetic complete medium without uracil and leucine. Plates were incubated at the indicated temperatures for 3 days. Images are representative of three experiments with duplicate plates for each temperature. Cue1p recruits Ubc7p to the surface of the ER and is necessary for ERAD function. By increasing the local Ubc7p concentration at the ER, Cue1p is thought to promote Ubc7p engagement with the ER-localized ERAD E3s. Our in vitro studies of membrane-anchored Hrd1p reveal that Cue1p reduces the concentration of Ubc7p required to observe ubiquitination (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Observations that soluble Cue1p lacking a transmembrane span (Cue1pΔ™) binds tightly to Ubc7p in vitro and causes cytosolic localization of GFP-Ubc7p in vivo (23Ravid T. Hochstrasser M. Nat. Cell Biol. 2007; 9: 422-427Crossref PubMed Scopus (136) Google Scholar) are also consistent with the model of Cue1p as anchor for Ubc7p. However, our previous in vitro studies of soluble, membrane-free Hrd1p function with Ubc7p suggested increased Ubc7p activity in the presence of Cue1p (11Bazirgan O.A. Garza R.M. Hampton R.Y. J. Biol. Chem. 2006; 281: 38989-39001Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). We considered that Cue1p might be an activator of Ubc7p E2 activity. To explore this possibility, we directly tested Cue1p activation of Ubc7p in a soluble in vitro ubiquitination assay. 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