Advertisement

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

INVOLVEMENT OF ER-ASSOCIATED UBIQUITIN-CONJUGATING ENZYMES (E2s)*
  • Swati Tiwari
    Affiliations
    From the Laboratory of Immune Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152
    Search for articles by this author
  • Allan M. Weissman
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Laboratory of Immune Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152
    Search for articles by this author
  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF 296656, AF 296657, and AF 296658.
Open AccessPublished:May 11, 2001DOI:https://doi.org/10.1074/jbc.M007640200
      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.
      Ub
      ubiquitin
      ER
      endoplasmic reticulum
      ERAD
      endoplasmic reticulum associated degradation
      TCR
      T cell antigen receptor
      CPY
      carboxypeptidase Y
      PnK
      proteinase K
      HA
      hemagglutinin
      PBS
      phosphate-buffered saline
      GFP
      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) (
      • Ciechanover A.
      ). 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 (
      • Bonifacino J.S.
      • Weissman A.M.
      ). 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 (
      • Hampton R.Y.
      • Bhakta H.
      ). 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 (
      • Hampton R.Y.
      • Gardner R.G.
      • Rine J.
      ), and a differential dependence among ERAD substrates on yeast HRD genes was recently demonstrated (
      • Wilhovsky S.
      • Gardner R.
      • Hampton R.
      ).
      In yeast, a number of ERAD substrates are multiubiquitinated, examples include mutant forms of Sec61p (
      • Biederer T.
      • Volkwein C.
      • Sommer T.
      ) and carboxypeptidase Y (CPY*) (
      • Hiller M.M.
      • Finger A.
      • Schweiger M.
      • Wolf D.H.
      ) as well as HMGCoA-reductase (
      • Hampton R.Y.
      • Bhakta H.
      ). 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 (
      • Biederer T.
      • Volkwein C.
      • Sommer T.
      ,
      • Hiller M.M.
      • Finger A.
      • Schweiger M.
      • Wolf D.H.
      ,
      • Plemper R.K.
      • Egner R.
      • Kuchler K.
      • Wolf D.H.
      ,
      • Galan J.M.
      • Cantegrit B.
      • Garnier C.
      • Namy O.
      • Haguenauer-Tsapis R.
      ). UBC6p is a C-terminal anchored membrane protein whose catalytic site faces the cytosol (
      • Sommer T.
      • Jentsch S.
      ). Unlike UBC6p, UBC7p lacks a membrane anchor but associates with an ER-bound protein, Cue1p (
      • Biederer T.
      • Volkwein C.
      • Sommer T.
      ).
      In mammalian cells ERAD substrates such as cystic fibrosis transmembrane conductance regulator and apoB are ubiquitinated in a cotranslational fashion in vitro (
      • Sato S.
      • Ward C.L.
      • Kopito R.R.
      ,
      • Zhou M.
      • Fisher E.A.
      • Ginsberg H.N.
      ). 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 (
      • Yang M.
      • Omura S.
      • Bonifacino J.S.
      • Weissman A.M.
      ). 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 (
      • Shamu C.E.
      • Story C.M.
      • Rapoport T.A.
      • Ploegh H.L.
      ). 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.

      DISCUSSION

      This study provides evidence that two mammalian E2s localize to the ER membrane. For MmUBC6 its C-terminal hydrophobic domain provides a basis for its membrane insertion. MmUBC7 lacks a membrane anchor that would allow for direct membrane insertion. In yeast, interaction with a C-terminal anchored protein, Cue1p, provides a molecular explanation for ER localization of UBC7p (
      • Biederer T.
      • Volkwein C.
      • Sommer T.
      ). Although mammalian Cue1p homologs have not been reported, it seems likely that an analogous protein may play a role in tethering of MmUBC7 to the ER membrane.
      Previous studies in cells expressing a temperature-sensitive ubiquitin activating enzyme (E1) have shown that a functional Ub pathway is required for degradation of TCR-α from the ER (
      • Yu H.
      • Kopito R.R.
      ). Data presented herein provides the first evidence implicating a specific E2, MmUBC7, in degradation from the ER in mammalian cells. Overexpression of catalytically inactive MmUBC7 results in decreased degradation of both the TCR-α and the CD3-δ subunits of the TCR. In contrast there is no evidence of a role for MmUBC6 in degradation of TCR-α. Similarly, no effect of mutant MmUBC6 was observed on degradation of CD3-δ.
      S. Tiwari and A. M. Weissman, unpublished observations.
      The negative data obtained with inactive MmUBC6 is consistent with deletion analyses in yeast where UBC7p is the predominant E2 in ERAD, whereas UBC6p has partial effect on degradation of certain proteins, including Sec61 (
      • Biederer T.
      • Volkwein C.
      • Sommer T.
      ) and CPY* (
      • Hiller M.M.
      • Finger A.
      • Schweiger M.
      • Wolf D.H.
      ), and no effect on Vph1 (
      • Hill K.
      • Cooper A.A.
      ).
      For proteins such as CD3-δ, which have cytoplasmically disposed lysines, it is easy to envisage models for ERAD that include ubiquitination of cytoplasmic lysines, recognition of ubiquitinated species by proteasomes, and dislocation and destruction from the ER facilitated by chaperone-like functions of proteasome. The lack of discernable retrotranslocation observed for CD3-δ in the presence of lactacystin with or without coexpression of inactive MmUBC7 is consistent with such a model and extends previous observations from our laboratory made on endogenous CD3-δ in T cells. Less obvious is how components of the Ub-conjugating system function in the retrotranslocation of lumenal proteins and of transmembrane proteins lacking cytoplasmic sites for ubiquitination, such as TCR-α. The N terminus of this protein is in the ER lumen, and it has no lysines in its cytoplasmic tail. When evaluated by pulse-chase analyses in T cells, TCR-α undergoes a discrete degree of proteasome-independent retrograde translocation that should allow exposure of potential sites of ubiquitination (
      • Yang M.
      • Omura S.
      • Bonifacino J.S.
      • Weissman A.M.
      ). However, similar evidence for partial retrograde movement is not obvious in our experiments in HEK-293 cells. Studies from other groups carried out in non-T cells have provided evidence that when expressed ectopically some level of complete retrograde translocation and accompanying deglycosylation of TCR-α occurs in the absence of proteasome function (
      • Yu H.
      • Kaung G.
      • Kobayashi S.
      • Kopito R.R.
      ,
      • Huppa J.B.
      • Ploegh H.L.
      ). Our observations corroborate these findings. However, even after 16 h of proteasome inhibition, the amount of cytoplasmically disposed TCR-α represents only a small fraction of the total accumulated material, suggesting a continued requirement for proteasome function for efficient complete retrotranslocation and degradation from the ER, as has been suggested for an engineered model substrate (
      • Mayer T.U.
      • Braun T.
      • Jentsch S.
      ) and for Pdr5 (
      • Plemper R.K.
      • Egner R.
      • Kuchler K.
      • Wolf D.H.
      ) in yeast. Similarly, a requirement for proteasome function in retrotranslocation has been reported for unassembled soluble Ig subunits (
      • Chillaron J.
      • Haas I.G.
      ) and CD4 (
      • Schubert U.
      • Anton L.C.
      • Bacik I.
      • Cox J.H.
      • Bour S.
      • Bennink J.R.
      • Orlowski M.
      • Strebel K.
      • Yewdell J.W.
      ,
      • Mancini R.
      • Fagioli C.
      • Fra A.M.
      • Maggioni C.
      • Sitia R.
      ) in mammalian cells. Results with TCR-α demonstrate that the relative amounts of cytoplasmically disposed material is consistently decreased when inactive MmUBC7 is coexpressed, even when proteasome function is inhibited, suggesting that MmUBC7 may play a role in the process leading to retrotranslocation upstream of the involvement of proteasomes. For CD3-δ the absence of detectable cytoplasmic forms under any circumstances precludes statements as to whether MmUBC7 act upstream of the proteasome. But it is evident that, as with TCR-α, this E2 is affecting the fate of CD3-δ prior to its removal from the ER membrane. Results obtained with both of these transmembrane TCR components are consistent with findings in yeast for a nontransmembrane ER protein, CPY*, where deletion of UBC7 resulted in its accumulation in the ER lumen (
      • Bordallo J.
      • Plemper R.K.
      • Finger A.
      • Wolf D.H.
      ).
      An important question is whether it is the ubiquitination of TCR-α itself by mammalian UBC7 homologs that is required for retrograde movement. Although this is the simplest explanation, and TCR-α clearly is a substrate for ubiquitination (
      • Yang M.
      • Omura S.
      • Bonifacino J.S.
      • Weissman A.M.
      ), the fact that TCR-α does not include cytoplasmic primary amines that could serve as sites of ubiquitination argues against this. Also against such a model is the finding that accumulation of retrotranslocated forms of lysine-less TCR-α in response to lactacystin is also inhibited by catalytically inactive MmUBC7. This therefore leads to the consideration of more complex models in which MmUBC7 enhances TCR-α loss indirectly, perhaps by ubiquitination of another protein that then facilitates translocation of TCR-α out of the ER. Applying such a model to other ERAD substrates would resolve the topological conundrum implicit in postulating ubiquitination of lumenal proteins such as CPY* as a primary event allowing for retrograde movement from the ER (
      • Bordallo J.
      • Plemper R.K.
      • Finger A.
      • Wolf D.H.
      ).
      Another issue that arises is the nature of the E3s with which MmUBC7 interacts. In yeast HRD1/DER3 is implicated in epistasis analysis along the same pathway as UBC7 in the degradation of several yeast proteins including CPY* and HMGCoA reductase (
      • Wilhovsky S.
      • Gardner R.
      • Hampton R.
      ). Whether mammalian homologs of HRD1 exist remains to be determined. Another ubiquitin ligase component implicated in degradation from the ER is the F-box protein βTRCP. This protein forms part of an SCF E3 complex and is implicated in the degradation of CD4 from the ER with phosphorylated HIV-1 Vpu functioning as an adaptor (
      • Schubert U.
      • Anton L.C.
      • Bacik I.
      • Cox J.H.
      • Bour S.
      • Bennink J.R.
      • Orlowski M.
      • Strebel K.
      • Yewdell J.W.
      ,
      • Margottin F.
      • Bour S.P.
      • Durand H.
      • Selig L.
      • Benichou S.
      • Richard V.
      • Thomas D.
      • Strebel K.
      • Benarous R.
      ). At least for nonmembrane proteins, SCF complex components are known to function with the yeast E2 CDC34 and its mammalian homologs. Whether βTRCP-containing SCF takes advantage of ER membrane-bound MmUBC7 or perhaps MmUBC6 when targeting CD4 for degradation from the ER now awaits determination.

      Acknowledgments

      We are grateful to Dr. Jim McNally for assistance with confocal microscopy, Dr. Juan Bonifacino and Dr. Ron Kopito for generously providing us with TCR-α and lysineless-TCR-α, respectively, Dr. Mei Yang for help in generating HA-tagged CD3-δ, and Dr. Lawrence Samelson for anti-CD3-δ. We thank Drs. Alessandra Magnifico, Shengyun Fang, Kevin Lorick, and Jane Jensen for helpful discussions and critical reading of the manuscript.

      REFERENCES

        • Ciechanover A.
        EMBO J. 1998; 17: 7151-7160
        • Bonifacino J.S.
        • Weissman A.M.
        Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57
        • Hampton R.Y.
        • Bhakta H.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948
        • Hampton R.Y.
        • Gardner R.G.
        • Rine J.
        Mol. Biol. Cell. 1996; 7: 2029-2044
        • Wilhovsky S.
        • Gardner R.
        • Hampton R.
        Mol. Biol. Cell. 2000; 11: 1697-1708
        • Biederer T.
        • Volkwein C.
        • Sommer T.
        EMBO J. 1996; 15: 2069-2076
        • Hiller M.M.
        • Finger A.
        • Schweiger M.
        • Wolf D.H.
        Science. 1996; 273: 1725-1728
        • Plemper R.K.
        • Egner R.
        • Kuchler K.
        • Wolf D.H.
        J. Biol. Chem. 1998; 273: 32848-32856
        • Galan J.M.
        • Cantegrit B.
        • Garnier C.
        • Namy O.
        • Haguenauer-Tsapis R.
        FASEB. J. 1998; 12: 315-323
        • Sommer T.
        • Jentsch S.
        Nature. 1993; 365: 176-179
        • Biederer T.
        • Volkwein C.
        • Sommer T.
        Science. 1997; 278: 1806-1809
        • Sato S.
        • Ward C.L.
        • Kopito R.R.
        J. Biol. Chem. 1998; 273: 7189-7192
        • Zhou M.
        • Fisher E.A.
        • Ginsberg H.N.
        J. Biol. Chem. 1998; 273: 24649-24653
        • Yang M.
        • Omura S.
        • Bonifacino J.S.
        • Weissman A.M.
        J. Exp. Med. 1998; 187: 835-846
        • Shamu C.E.
        • Story C.M.
        • Rapoport T.A.
        • Ploegh H.L.
        J. Cell Biol. 1999; 147: 45-58
        • Pari G.S.
        • Keown W.A.
        Methods Mol. Biol. 1997; 62: 301-306
        • Samelson L.E.
        • Weissman A.M.
        • Robey F.A.
        • Berkower I.
        • Klausner R.D.
        J. Immunol. 1986; 137: 3254-3258
        • Cenciarelli C.
        • Wilhelm K.G.J.
        • Guo A.
        • Weissman A.M.
        J. Biol. Chem. 1996; 271: 8709-8713
        • Kubo R.T.
        • Born W.
        • Kappler J.W.
        • Marrack P.
        • Pigeon M.
        J. Immunol. 1989; 142: 2736-2742
        • Guan K.L.
        • Dixon J.E.
        Anal. Biochem. 1991; 192: 262-267
        • Kozak M.
        J. Cell Biol. 1989; 108: 229-241
        • Yang M.
        • Ellenberg J.
        • Bonifacino J.S.
        • Weissman A.M.
        J. Biol. Chem. 1997; 272: 1970-1975
        • Haas A.L.
        • Siepmann T.J.
        FASEB J. 1997; 11: 1257-1268
        • Katsanis N.
        • Fisher E.M.
        Genomics. 1998; 51: 128-131
        • Lin H.
        • Wing S.S.
        J. Biol. Chem. 1999; 274: 14685-14691
        • Jensen J.P.
        • Bates P.W.
        • Yang M.
        • Vierstra R.D.
        • Weissman A.M.
        J. Biol. Chem. 1995; 270: 30408-30414
        • Yu H.
        • Kopito R.R.
        J. Biol. Chem. 1999; 274: 36852-36858
        • Hill K.
        • Cooper A.A.
        EMBO J. 2000; 19: 550-561
        • Yu H.
        • Kaung G.
        • Kobayashi S.
        • Kopito R.R.
        J. Biol. Chem. 1997; 272: 20800-20804
        • Huppa J.B.
        • Ploegh H.L.
        Immunity. 1997; 7: 113-122
        • Mayer T.U.
        • Braun T.
        • Jentsch S.
        EMBO J. 1998; 17: 3251-3257
        • Chillaron J.
        • Haas I.G.
        Mol. Biol. Cell. 2000; 11: 217-226
        • Schubert U.
        • Anton L.C.
        • Bacik I.
        • Cox J.H.
        • Bour S.
        • Bennink J.R.
        • Orlowski M.
        • Strebel K.
        • Yewdell J.W.
        J. Virol. 1998; 72: 2280-2288
        • Mancini R.
        • Fagioli C.
        • Fra A.M.
        • Maggioni C.
        • Sitia R.
        FASEB J. 2000; 14: 769-778
        • Bordallo J.
        • Plemper R.K.
        • Finger A.
        • Wolf D.H.
        Mol. Biol. Cell. 1998; 9: 209-222
        • Margottin F.
        • Bour S.P.
        • Durand H.
        • Selig L.
        • Benichou S.
        • Richard V.
        • Thomas D.
        • Strebel K.
        • Benarous R.
        Mol Cell. 1998; 1: 565-574