Endoplasmic Reticulum (ER)-associated Degradation of T Cell Receptor Subunits

Degradation of proteins from the endoplasmic reticulum is fundamental to quality control within the secretory pathway, serves as a way of regulating levels of crucial proteins, and is utilized by viruses to enhance pathogenesis. In yeast two ubiquitin-conjugating enzymes (E2s), UBC6p and UBC7p are implicated in this process. We now report the characterization of murine homologs of these E2s. MmUBC6 is an integral membrane protein that is anchored via its hydrophobic C-terminal tail to the endoplasmic reticulum. MmUBC7, which is not an integral membrane protein, shows significant endoplasmic reticulum colocalization with MmUBC6. Overexpression of catalytically inactive MmUBC7 significantly delayed degradation from the endoplasmic reticulum of two T cell antigen receptor subunits, α and CD3-δ, and suggests a role for the ubiquitin conjugating system at the initiation of retrograde movement from the endoplasmic reticulum. These findings also implicate, for the first time, a specific E2 in degradation from the endoplasmic reticulum in mammalian cells. Degradation of proteins from the endoplasmic reticulum is fundamental to quality control within the secretory pathway, serves as a way of regulating levels of crucial proteins, and is utilized by viruses to enhance pathogenesis. In yeast two ubiquitin-conjugating enzymes (E2s), UBC6p and UBC7p are implicated in this process. We now report the characterization of murine homologs of these E2s. MmUBC6 is an integral membrane protein that is anchored via its hydrophobic C-terminal tail to the endoplasmic reticulum. MmUBC7, which is not an integral membrane protein, shows significant endoplasmic reticulum colocalization with MmUBC6. Overexpression of catalytically inactive MmUBC7 significantly delayed degradation from the endoplasmic reticulum of two T cell antigen receptor subunits, α and CD3-δ, and suggests a role for the ubiquitin conjugating system at the initiation of retrograde movement from the endoplasmic reticulum. These findings also implicate, for the first time, a specific E2 in degradation from the endoplasmic reticulum in mammalian cells. ubiquitin endoplasmic reticulum endoplasmic reticulum associated degradation T cell antigen receptor carboxypeptidase Y proteinase K hemagglutinin phosphate-buffered saline green fluorescent protein In eukaryotes, a primary means by which proteins are targeted for degradation is by their modification with chains of ubiquitin (Ub).1 Ubiquitinated proteins are recognized and degraded by the multicatalytic 26 S proteasome. Attachment of Ub to proteins involves a process in which one of a number of different Ub-conjugating enzymes (UBCs or E2s) accept Ub from activated E1 enzyme in a transthiolation reaction and subsequently catalyze the formation of an isopeptide bond between Ub and substrate, either with or without the involvement of an Ub-protein ligase (E3) (1Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1200) Google Scholar). Proteasomal degradation is not limited to proteins native to the nucleus and cytosol where proteasomes reside. Many transmembrane and lumenal proteins of the secretory pathway are degraded from the endoplasmic reticulum (ER) by proteasomes. The processes that ultimately result in the proteasomal degradation of these proteins are referred to as ERAD (ER-associated degradation). Steps involved in ERAD can include trimming of N-linked glycans, ubiquitination, retrograde movement through the ER membrane, deglycosylation, and degradation in the cytosol by proteasomes (2Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar). The temporal and mechanistic relationships between retrograde movement, conjugation with Ub, and possible chaperone-like functions of proteasomes appear to differ based on the nature of the substrate. This might be expected given the varied substrates, which include luminal proteins, proteins having a single membrane-spanning domain, and complex polytopic proteins. Moreover, these substrates may be either mutated misfolded proteins, otherwise normal proteins that have failed to assemble in a complex, or, as is the case with HMGCoA reductase, a normal protein whose activity is regulated by ERAD (3Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar). Consistent with different requirements for degradation of these varied substrates, a genetic analysis of yeast mutants that are defective in ERAD of HMG-CoA reductase led to the identification of HRD genes (4Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (466) Google Scholar), and a differential dependence among ERAD substrates on yeast HRD genes was recently demonstrated (5Wilhovsky S. Gardner R. Hampton R. Mol. Biol. Cell. 2000; 11: 1697-1708Crossref PubMed Scopus (95) Google Scholar). In yeast, a number of ERAD substrates are multiubiquitinated, examples include mutant forms of Sec61p (6Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar) and carboxypeptidase Y (CPY*) (7Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar) as well as HMGCoA-reductase (3Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (121) Google Scholar). Genetic analysis has implicated two yeast E2s, UBC6p and UBC7p, in ERAD. Deletion of UBC6 and UBC7 stabilizes mutant Sec61p, Sss1p, CPY, Pdr5, and uracil permease (6Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar, 7Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar, 8Plemper 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, 9Galan J.M. Cantegrit B. Garnier C. Namy O. Haguenauer-Tsapis R. FASEB. J. 1998; 12: 315-323Crossref PubMed Scopus (25) Google Scholar). UBC6p is a C-terminal anchored membrane protein whose catalytic site faces the cytosol (10Sommer T. Jentsch S. Nature. 1993; 365: 176-179Crossref PubMed Scopus (283) Google Scholar). Unlike UBC6p, UBC7p lacks a membrane anchor but associates with an ER-bound protein, Cue1p (11Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar). In mammalian cells ERAD substrates such as cystic fibrosis transmembrane conductance regulator and apoB are ubiquitinated in a cotranslational fashion in vitro (12Sato S. Ward C.L. Kopito R.R. J. Biol. Chem. 1998; 273: 7189-7192Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 13Zhou M. Fisher E.A. Ginsberg H.N. J. Biol. Chem. 1998; 273: 24649-24653Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Subunits of the T cell antigen receptor (TCR), when not assembled into complexes capable of exiting the ER, are also degraded from the ER. In T lymphocytes multiubiquitinated forms of TCR-α and the TCR CD3-δ subunit are associated with the ER membrane, suggesting that their ubiquitination occurs while still membrane-bound (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). In initial studies on ERAD of major histocompatability complex class I proteins, evidence for ubiquitination was lacking. However, more recent analyses have provided evidence for ubiquitinated major histocompatability complex class I molecules as degradation intermediates (15Shamu C.E. Story C.M. Rapoport T.A. Ploegh H.L. J. Cell Biol. 1999; 147: 45-58Crossref PubMed Scopus (130) Google Scholar). Collectively, these finding suggest that in mammals, as in yeast, components of the Ub conjugating machinery functionally interact with substrates at the ER membrane. Despite a clear role for ERAD in mammals, no specific E2s have been implicated in this process. We now report characterization of mammalian E2s homologous to yeast UBC6p and UBC7p, establish that these proteins are ER membrane proteins, and provide evidence that a murine UBC7p homolog, MmUBC7, plays a role in the degradation of unassembled TCR subunits from the ER. Cos-7 (number CRL1651; American Tissue Culture Collection, Manassas, VA) and HEK-293 (number CRL1573; American Tissue Culture Collection) cells were maintained in complete Dulbecco's modified Eagle's medium and transfected using the calcium-phosphate method as described (16Pari G.S. Keown W.A. Methods Mol. Biol. 1997; 62: 301-306PubMed Google Scholar). Anti-CD3-δ (R9) (17Samelson L.E. Weissman A.M. Robey F.A. Berkower I. Klausner R.D. J. Immunol. 1986; 137: 3254-3258PubMed Google Scholar); anti-ubiquitin (18Cenciarelli C. Wilhelm K.G.J. Guo A. Weissman A.M. J. Biol. Chem. 1996; 271: 8709-8713Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar); and anti-TCR-α, H28 (19Kubo R.T. Born W. Kappler J.W. Marrack P. Pigeon M. J. Immunol. 1989; 142: 2736-2742PubMed Google Scholar) have all been described. Anti-HA (12CA5) and anti-Myc (9E10) monoclonal antibodies were from culture supernatants. Anti-GRP78 (BiP) antibody was fromStressGen Biotech. (Victoria, Canada). Anti-MDM2 was from Oncogene Science (Cambridge, MA). Lysineless TCR-α (K→RTCR-α/pcDNA3.1) was a gift from Dr. Ron Kopito. Wild type 2B4 TCR-α in pCDM8 (Invitrogen, Carlsbad, CA) and CD3-δ in pCI (Promega, Madison, WI) were obtained from Dr. Juan Bonifacino. MDM2/pCINeo was a gift from Dr. Shengyun Fang. GenBankTM expressed sequence tag data bases were searched using the amino acid sequences of yeast UBC6p and UBC7p. Murine cDNA clones that were homologous to yeast sequences (referred to as MmUBC6 and MmUBC7) were obtained and sequenced. MmUBC6 was cloned into pCI vector and tagged with HA epitope at the N-terminal by polymerase chain reaction using the primers 5′-ATAGAATTCACCATGGCCTACCCATACGACGTCCCAGACTACGCTCCCGGGCCCGAGATTAGCAATAAC-3′ and 5′-GCCACCTTCATAAGGAGTCATC-3′. The polymerase chain reaction product was digested with EcoRI and SacI and cloned into pCI digested with the same enzymes. The construct was confirmed by sequencing. To generate MmUBC6 lacking the C-terminal tail, nucleotides encoding the last 52 amino acids were removed by restriction enzyme digestion, and the remainder of the cDNA was subcloned into pCDNA3 (Invitrogen, Carlsbad, CA). MmUBC7 was tagged with Myc epitope at the N terminus using primers 5′-ATATGAATTCATGGAGCAGAAGCTGATTTCCGAGGAGGACCTGAACCTCAAATTGGCGGGGACGGCGTTGAAG-3′ and 5′-AACGACGGCCAGTGCCAAGC-3′. The polymerase chain reaction product was digested with EcoRI and NotI and cloned into pcDNA3. The construct was confirmed by sequencing. Site-directed point mutations were created using QuickChange Mutagenesis kit (Stratagene, La Jolla, CA). The mutations were confirmed by sequencing. MmUBC6 and MmUBC7 were also cloned into pGEX-KG (20Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar) to generate N-terminal glutathione S-transferase fusions. For some experiments, glutathione S-transferase moieties were cleaved by thrombin treatment and residual thrombin was removed by to the in vitro and was in (Promega, Madison, WI) in the or of were by at for in an For the were in for at by and of and membrane. The was with acid and the in Cos-7 cells were on and transfected using CA) to were with in phosphate-buffered saline for with This was by with or antibody in and for by with and with antibody to For were with primary antibody to by with to were obtained on a using and The of the and of the were to and was between and for and between and for HEK-293 cells were transfected using calcium-phosphate method with of 2B4 and of either type or mutant or or The of was with were and for in medium and for with of CA). were at with complete medium by in complete medium at were at and were as described by and were in as described (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). were at using protein to were with In some was in the and were to were using either protein or were in and as described (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). cells and were removed by at for The was in and one was with proteinase K as described (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar) were at for at were with and in To the was were at for and the was with H28 with was as by the To mammalian of yeast UBC6p, the GenBankTM expressed sequence tag data was searched and homologous murine obtained and sequenced. such a that was in a for initiation M. J. Cell Biol. 1989; PubMed Scopus Google Scholar) with a to this site in the same number The a protein of amino amino acids more UBC6p This protein is referred to as MmUBC6 for the murine of yeast UBC6p based on the that E2s are for the by a with UBC6p is in the the E2 acids of where is and However, amino acid of the protein is MmUBC6 was to be an E2 by the of MmUBC6 to with Ub in the of E1 not This activity was by of the site acid UBC6p is among E2s in that is a type membrane protein, as such by its C-terminal membrane anchor that for its ER membrane in yeast (10Sommer T. Jentsch S. Nature. 1993; 365: 176-179Crossref PubMed Scopus (283) Google Scholar) and when expressed in mammalian cells M. J. Bonifacino J.S. Weissman A.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). MmUBC6 has a C-terminal hydrophobic domain, is from yeast analysis using and Madison, WI) suggests that this at amino acid and at to amino acid and to amino acid which to UBC6p, also shows in the C-terminal hydrophobic However, a expressed sequence tag homologous to yeast UBC6p was also and was to be to MmUBC6 with a single amino acid in the C-terminal hydrophobic number This be referred to as encoding be that is not a of the mammalian of E2 enzymes J. 1997; 11: PubMed Scopus Google Scholar). To MmUBC6 is a membrane protein, was in in the of This a protein of which more was in the membrane and MmUBC6 was to and and into the with and lacking the C-terminal hydrophobic into the MmUBC6 is also a C-terminal integral membrane C-terminal anchored membrane proteins have been to be in To this is the case for was in vitro without and for membrane by with by of membrane and by were of MmUBC6 into the membrane and and was to and In was in the expressed in mammalian the C-terminal hydrophobic of yeast UBC6p from to amino acids in a the secretory pathway from the ER to the M. J. Bonifacino J.S. Weissman A.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The hydrophobic of MmUBC6 is its yeast However, its by a ER and colocalization with UBC6p MmUBC6 is also an ER membrane of MmUBC6 and Cos-7 cells were transfected with and and control cells with and anti-Myc of MmUBC6 cell with MmUBC6 and is in where MmUBC6 is in is in and the of is of the two proteins is and homologs of UBC7p have been Fisher 1998; PubMed Scopus Google Scholar, H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar); no evidence for or with the ER has been murine UBC7p was in the expressed sequence tag data that an a protein of amino acids that and with yeast UBC7p, and and with its number We to this protein as In vitro in an protein, with the amino acid The of this E2 to with Ub was confirmed not MmUBC7 lacks a hydrophobic and is to be a However, of MmUBC7 with in vitro in of MmUBC7 membrane associated M. R.D. Weissman A.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) not to of the membrane of MmUBC7 was by of MmUBC7 was in the and membrane and In all of the was in the by MmUBC7 to be but a ER is To this ER was with MmUBC6. shows from the same cell and significant colocalization of with to the ER was when and were shows studies have that ERAD of a type I transmembrane protein with a tail of amino is on To mammalian homologs of UBC6p and UBC7p are involved in TCR-α for catalytically inactive forms of these E2s were with TCR-α in HEK-293 cells. of TCR-α were by with of a mutant of MmUBC6 in which the site was to not TCR-α the catalytically inactive MmUBC7 in in TCR-α levels and of the two inactive E2s not result in a In of inactive MmUBC7 not result in of which is a protein that is ubiquitinated and degraded by proteasomes type MmUBC6 MmUBC7 significant or on TCR-α To that the with of was to of TCR-α was by TCR-α a in when was of inactive MmUBC6 not TCR-α of TCR-α in HEK-293 cells from of in a studies in cells have that when is a significant of the TCR-α has from the ER to the cytosol by H. S. Kopito R.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Ploegh H.L. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). To the of TCR-α that with inactive MmUBC7, cells TCR-α were to proteinase K by of and membrane inactive MmUBC7 was of TCR-α were and of this TCR-α was in the membrane and was to and has not through the ER membrane. was and levels of TCR-α still the of the was in the membrane to However, a of the TCR-α was and and more that forms and in the as well as in the membrane these forms were in all when with the levels of forms were at a in cells inactive The findings that inactive MmUBC7 in the of TCR-α and that is a in when is MmUBC7 in a role in ERAD to retrograde movement through the ER membrane. the when is are with for TCR-α where of N-linked is with to the Consistent with TCR-α with with the forms of TCR-α To the on TCR-α degradation with are on ubiquitination of TCR-α on a of TCR-α H. Kopito R.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This of TCR-α is degraded from the ER in a a of for is TCR-α also when inactive MmUBC7 is with in of with type of these forms is when is as be these were not The CD3-δ subunit of the TCR is also a for ERAD when to assemble with TCR components and the ER (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). This protein from TCR-α in having a and a single in its transmembrane To MmUBC7 is also involved in the degradation of this protein, analysis of CD3-δ was CD3-δ was expressed in HEK-293 was degraded However, as with its was significantly when with catalytically inactive MmUBC6 not significantly CD3-δ degradation To CD3-δ a C-terminal was by that was degraded with from type CD3-δ not was to the of catalytically inactive MmUBC7 on levels of CD3-δ by levels of CD3-δ either when catalytically inactive MmUBC7 was or when was Unlike for which forms are when is studies in T cells not evidence of of CD3-δ (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Consistent with of or not inactive MmUBC7 was with CD3-δ, forms that in the of were limited in to the membrane These are in with the of membrane and proteasomal activity for CD3-δ and suggest as with MmUBC7 is to retrograde movement through the ER membrane This evidence that two mammalian E2s to the ER membrane. For MmUBC6 its C-terminal hydrophobic a for its membrane MmUBC7 lacks a membrane anchor that for membrane In yeast, with a C-terminal anchored protein, a for ER of UBC7p (11Biederer T. Volkwein C. Sommer T. Science. 1997; 278: 1806-1809Crossref PubMed Scopus (331) Google Scholar). mammalian Cue1p homologs have not been that an protein may a role in of MmUBC7 to the ER membrane. studies in cells a ubiquitin enzyme have that a Ub pathway is for degradation of TCR-α from the ER H. Kopito R.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the first evidence a specific MmUBC7, in degradation from the ER in mammalian cells. Overexpression of catalytically inactive MmUBC7 in degradation of the TCR-α and the CD3-δ subunits of the In is no evidence of a role for MmUBC6 in degradation of no of mutant MmUBC6 was on degradation of and A. M. The data obtained with inactive MmUBC6 is with analyses in yeast where UBC7p is the E2 in UBC6p has on degradation of proteins, (6Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar) and (7Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar), and no on K. EMBO J. 2000; PubMed Scopus Google Scholar). For proteins such as CD3-δ, which have is to for ERAD that include ubiquitination of of ubiquitinated by and and from the ER by chaperone-like functions of proteasome. The of for CD3-δ in the of with or without of inactive MmUBC7 is with such a and from on CD3-δ in T cells. is components of the Ub-conjugating system in the of lumenal proteins and of transmembrane proteins lacking for ubiquitination, such as The N terminus of this protein is in the ER and has no in its by analyses in T TCR-α a of retrograde that of of ubiquitination (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). However, evidence for retrograde movement is not in in HEK-293 cells. from in cells have provided evidence that when expressed some of complete retrograde and of TCR-α occurs in the of H. S. Kopito R.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Ploegh H.L. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). these However, of the of TCR-α a of the suggesting a for for complete and degradation from the ER, as has been for an T. Jentsch S. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and for 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) in a for in has been for unassembled subunits J. Mol. Biol. Cell. 2000; 11: PubMed Scopus Google Scholar) and U. I. S. M. K. J.W. J. 1998; PubMed Google Scholar, R. C. A.M. C. R. J. 2000; 14: PubMed Scopus Google Scholar) in mammalian cells. with TCR-α that the of is when inactive MmUBC7 is when is suggesting that MmUBC7 may a role in the process to of the involvement of proteasomes. For CD3-δ the of forms as to MmUBC7 of the proteasome. is as with this E2 is the of CD3-δ to its from the ER membrane. obtained with of these transmembrane TCR components are with findings in yeast for a ER protein, where of UBC7 in its in the ER J. R.K. Finger A. Wolf D.H. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar). is is the ubiquitination of TCR-α by mammalian UBC7 homologs that is for retrograde this is the and TCR-α is a for ubiquitination (14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar), the that TCR-α not include primary that as of ubiquitination such a is the finding that of forms of TCR-α in to is also by catalytically inactive This to the of more complex in which MmUBC7 TCR-α by ubiquitination of protein that of TCR-α of the ER. such a to ERAD substrates the in ubiquitination of lumenal proteins such as as a primary for retrograde movement from the ER J. R.K. Finger A. Wolf D.H. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar). that is the nature of the with which MmUBC7 In yeast is implicated in analysis the same pathway as UBC7 in the degradation of yeast proteins and HMGCoA reductase (5Wilhovsky S. Gardner R. Hampton R. Mol. Biol. Cell. 2000; 11: 1697-1708Crossref PubMed Scopus (95) Google Scholar). mammalian homologs of to be ubiquitin ligase implicated in degradation from the ER is the protein This protein forms of an complex and is implicated in the degradation of from the ER with as an U. I. S. M. K. J.W. J. 1998; PubMed Google Scholar, H. S. K. R. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). for proteins, complex components are to with the yeast E2 and its mammalian of ER membrane-bound MmUBC7 or MmUBC6 when for degradation from the ER now We are to Dr. for with Dr. Juan Bonifacino and Dr. Ron Kopito for with TCR-α and Dr. for in CD3-δ, and Dr. for We Shengyun and for and of the

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