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Calnexin Association Is Not Sufficient to Protect T Cell Receptor α Proteins from Rapid Degradation in CD4+CD8+ Thymocytes

1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês

10.1074/jbc.273.37.23674

1083-351X

Michael J. Bennett, Jeroen E.M. van Leeuwen, Kelly P. Kearse,

Heme Oxygenase-1 and Carbon Monoxide

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRα molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRα instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRα proteins in all other T cell types examined. Here, we compared the processing of TCRα glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRα glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRα proteins stably associated with calnexin in both cell types. Interestingly, however, TCRα association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+thymocytes compared with splenic T cells. Finally, TCRα degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin·TCRα complexes. These data demonstrate that calnexin association is not sufficient to protect TCRα proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRα glycoproteins. During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRα molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRα instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRα proteins in all other T cell types examined. Here, we compared the processing of TCRα glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRα glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRα proteins stably associated with calnexin in both cell types. Interestingly, however, TCRα association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+thymocytes compared with splenic T cells. Finally, TCRα degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin·TCRα complexes. These data demonstrate that calnexin association is not sufficient to protect TCRα proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRα glycoproteins. endoplasmic reticulum T cell receptor antibody monoclonal Ab N-tosyl-l-phenylalanine chloromethyl ketone endoglycosidase H polyacrylamide gel electrophoresis glucose castanospermine. Coincident with their synthesis and translocation into the lumen of the endoplasmic reticulum (ER),1 many newly synthesized polypeptides are modified by the addition of oligosaccharide chains, having the structure Glc3Man9GlcNAc2 (Glc = glucose; Man = mannose; GlcNAc = N-acetyl glucosamine) (1Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3776) Google Scholar, 2Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar). Immature N-glycan chains are initially processed by the sequential action of glucosidase I and glucosidase II enzymes (the Glc-trimming pathway), creating monoglucosylated Glc1Man9GlcNAc2 glycans important for interaction with the molecular chaperones calnexin and calreticulin (3Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 4Spiro R.G. Zhu Q. Bhoyroo V. Soling H.-D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Calnexin and calreticulin are transmembrane and luminal proteins, respectively, that are proposed to function in the ER quality control system of protein folding and assembly (5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 6Peterson J.R. Ori A. Van P.N. Helenius A. Mol. Biol. Cell. 1995; 6: 1173-1184Crossref PubMed Scopus (266) Google Scholar, 7Otteken A. Moss B. J. Biol. Chem. 1996; 271: 97-103Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 8Nauseef W.M. McCormick S.J. Clark R.A. J. Biol. Chem. 1995; 270: 4741-4747Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 9Vassilakos A. Cohen-Doyle M.F. Peterson P.A. Jackson M.R. Williams D.B. EMBO J. 1996; 15: 1495-1506Crossref PubMed Scopus (170) Google Scholar, 10Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 11Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). Assembly of most newly synthesized glycoproteins with calnexin and calreticulin occurs by a two-step process involving initial recognition of monoglucosylated glycans, followed by protein-protein interactions, which stabilize these associations (3Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 11Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). The mechanisms important for release of proteins from calnexin and calreticulin are not well understood, although for certain molecules cleavage of the final (innermost) Glc residue by glucosidase II appears to be important (11Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 12Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (221) Google Scholar, 13van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 14van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (76) Google Scholar). Studies using glucosidase inhibitors and mutant cell lines deficient in expression of glucosidase enzymes have established a strong correlation between Glc trimming, calnexin association, and the survival of newly synthesized T cell receptor (TCR) α molecules in the ER (13van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Indeed, persistence of Glc residues on N-glycans precludes formation of calnexin·TCRα complexes and results in the rapid degradation of TCRα proteins (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar), occurring much faster than is typically observed for TCRα protein turnover under conditions where glycan processing is unperturbed. These results are specific in that the stability of related TCRβ glycoproteins is unaffected by impaired glucosidase activity and the failure to assemble with calnexin (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar, 19van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, calnexin association appears to be uniquely important for the survival of nascent TCRα glycoproteins in the ER. Detailed investigation into the production of TCR complexes in CD4+CD8+ thymocytes showed that inefficient assembly of complete αβδεγεζζ TCR results from limited assembly of αδε intermediates, which, in turn, results from rapid degradation of TCRα proteins (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 21Kearse K.P. Takahama Y. Punt J.A. Sharrow S.O. Singer A. J. Exp. Med. 1995; 181: 193-202Crossref PubMed Scopus (78) Google Scholar). Instability of TCRα proteins in CD4+CD8+ thymocytes is independent of clonotypic specificities as even a matched pair of TCRα, β transgenes fails to associate efficiently in CD4+CD8+ thymocytes (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar) and is developmentally regulated as TCRα proteins synthesized in immature CD4+CD8+ thymocytes have a significantly shorter half-life than those made in progeny CD4+ and CD8+ T cells (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 21Kearse K.P. Takahama Y. Punt J.A. Sharrow S.O. Singer A. J. Exp. Med. 1995; 181: 193-202Crossref PubMed Scopus (78) Google Scholar). The molecular basis for TCRα instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRα proteins in other T cell types (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar,18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar). To enhance our understanding of the molecular basis for the limited survival of newly synthesized TCRα proteins in immature CD4+CD8+ thymocytes, we undertook the present study to examine and compare the processing of TCRα glycoproteins and their assembly with lectin-like chaperones in CD4+CD8+ thymocytes and splenic T cells. C57BL/6 (B6) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). T cell receptor Vα11+ AND transgenic mice (22Kaye J. Hsu M.L. Sauron M.E. Jameson S.C. Gascoigne N.R. Hedrick S.M. Nature. 1989; 341: 746-749Crossref PubMed Scopus (589) Google Scholar) were generously provided by Dr. Stephen Hedrick. CD4+CD8+thymocytes were isolated by their adherence to plastic plates coated with anti-CD8 mAb (83-12-5) and were typically >95% CD4+CD8+ as described (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). Splenic T cells were purified by incubation of single cell suspensions of spleen cells on tissue culture plates coated with rabbit anti-mouse immunoglobulin (Ig) (Organo-Technika-Cappel, Malvern, PA) for 60 min at 37 °C, followed by isolation of nonadherent cells. The resultant cell populations were typically 80–85% CD3+ as determined by flow cytometry analysis. Castanospermine (Calbiochem) was used at a final concentration of 100 μg/ml. Diamide (Sigma) was used at a final concentration of 1 mm;N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) (Calbiochem) was used at a final concentration of 100 μg/ml;N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (Sigma) was used at a final concentration of 10 μg/ml; lactacystin (Calbiochem) was used at a final concentration of 20 μm. The following monoclonal antibodies (mAb) were used in this study: 145-2C11, specific for CD3ε proteins associated with CD3δ or CD3γ chains (23Leo O. Foo M. Sachs D.H. Samelson L.E. Bluestone J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1374-1378Crossref PubMed Scopus (1705) Google Scholar); H28-710, specific for TCRα (24Becker J.L.B. Near R. Mudgett-Hunter M. Margolies M.M. Kubo R.T. Kaye J. Hedrick S.M. Cell. 1989; 58: 911-921Abstract Full Text PDF PubMed Scopus (108) Google Scholar); H57-597, specific for TCRβ (25Kubo R.T. Born J.W. Kappler J. Marrack P. Pigeon M. J. Immunol. 1989; 142: 2736-2742PubMed Google Scholar); and anti-Vα11 Ab (Pharmingen, San Diego, CA). The following antisera were used: SPA-860 anti-calnexin (Stressgen Biotechnologies, Victoria, BC); and PA3–900 anti-calreticulin (Affinity BioReagents, Neshanic Station, NJ). Metabolic pulse labeling with [35S]methionine was performed as described previously (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). Briefly, cells were pulse-labeled in methionine-deficient medium containing [35S]-methionine (Trans 35S-label; ICN, Irvine, CA) for 30 min at 37 °C and chased in medium containing excess nonradioactive methionine for various time periods at 37 °C. Cells were lysed by solubilization in 1% digitonin (Calbiochem) lysis buffer (20 mm Tris, 150 mm NaCl, plus protease inhibitors) at 1 × 108 cells/ml for 20 min at 4 °C; lysates were clarified by centrifugation to remove insoluble material and immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads (Pharmacia, Uppsala, Sweden). Sequential immunoprecipitation and immunoprecipitation/release/recapture procedures were performed as previously detailed (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Digestion with endoglycosidase H (Endo H) was performed by resuspending precipitates in glycosidase digestion buffer (75 mm sodium phosphate, pH 6.1, 75 mm EDTA, 0.1% Nonidet P-40) containing 10 milliunits of Endo H (Genzyme, Cambridge, MA) for 16 h at 37 °C; digestion with jack bean mannosidase (Oxford Glycosystems, Rosedal, NY) was performed according to the manufacturer's instructions. For experiments examining the effects of oligosaccharide release on calnexin association with TCRα proteins, lysates were precipitated with anti-calnexin Abs and precipitates either mock-treated (buffer only) or digested with Endo H as described above. Precipitates were then washed 3 times with phosphate-buffered saline, boiled in 1% SDS, and TCRα proteins specifically recaptured with anti-TCRα mAbs as described (20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). SDS-PAGE gel electrophoresis was performed as previously detailed (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). Initially, we examined the assembly of newly synthesized glycoproteins with calnexin in CD4+CD8+ thymocytes. For these studies, CD4+CD8+ thymocytes were radiolabeled with [35S]methionine for 30 min, solubilized in 1% digitonin, and lysates immunoprecipitated with anti-calnexin Ab; alternatively, lysates were sequentially immunoprecipitated with anti-CD3ε mAb followed by anti-TCRα mAb. Precipitates were digested with Endo H glycosidase to identify molecules containing (immature)N-linked glycan chains (26Tarentino A.L. Maley F. J. Biol. Chem. 1974; 249: 811-816Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 1 A, numerous Endo H-sensitive proteins were associated with calnexin in CD4+CD8+ thymocytes (Fig. 1 A), including molecules that comigrated with TCRα and CD3δ glycoproteins (Fig. 1 A). Analysis of anti-calnexin precipitates on two-dimensional nonequilibrium pH gradient electrophoresis SDS-PAGE gels showed that numerous radiolabeled proteins assembled with calnexin in CD4+CD8+thymocytes (Fig. 1 B), most of which were not associated at the conclusion of a 60-min chase period (Fig. 1 B). Taken together, these data indicate that newly synthesized glycoproteins interact transiently with calnexin in CD4+CD8+thymocytes, similar to what has been reported regarding calnexin-protein associations in other cell types (5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 10Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 11Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 12Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (221) Google Scholar). Previous studies in splenic T cells showed that Glc residues are removed from newly synthesized TCRα glycoproteins before their assembly with CD3 subunits and that calnexin associates exclusively with unassembled, free TCRα proteins containing incompletely trimmed glycan chains (17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To determine whether TCRα proteins were equivalently processed in CD4+CD8+ thymocytes, similar experiments were performed; because TCRα proteins are clonotypic and quite heterogeneous, as in previous studies using splenic T cells (17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), CD4+CD8+ thymocytes were obtained from AND transgenic mice expressing the rearranged TCRα gene product, Vα11 protein (22Kaye J. Hsu M.L. Sauron M.E. Jameson S.C. Gascoigne N.R. Hedrick S.M. Nature. 1989; 341: 746-749Crossref PubMed Scopus (589) Google Scholar). CD4+CD8+ thymocytes were pulse-labeled with [35S]methionine for 30 min, solubilized in 1% digitonin, and lysates sequentially immunoprecipitated with anti-CD3ε mAb to isolate CD3ε-associated TCRα proteins, followed by anti-Vα11 mAb to capture unassembled TCRα chains. Alternatively, lysates were precipitated with anti-calnexin Ab. Precipitates were boiled in SDS to release bound material, TCRα proteins specifically recaptured by precipitation with anti-TCRα Ab, and recapture precipitates digested with jack bean mannosidase and Endo H glycosidases. Jack bean mannosidase digestion is useful for evaluating the Glc-trimming status of newly synthesized glycoproteins as it removes eight mannoses from fully trimmedN-glycan chains devoid of Glc residues (Man8–9GlcNAc2), but only five mannoses from incompletely trimmed N-glycans containing 1–3 Glc saccharides (Glc1–3Man8–9GlcNAc2) (5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In contrast, Endo H removes all but a single GlcNAc fromN-glycan chains irrespective of their Glc content (26Tarentino A.L. Maley F. J. Biol. Chem. 1974; 249: 811-816Abstract Full Text PDF PubMed Google Scholar). Similar to what was previously observed in splenic T cells (17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), two major glycoforms of Vα11 proteins were present in CD4+CD8+ thymocyte lysates, denoted A and B, that are indicative of TCRα proteins containing incompletely trimmed and fully trimmed glycan chains, respectively (Fig. 2). As demonstrated, Vα11 proteins that were assembled with CD3 subunits in CD4+CD8+thymocytes possessed fully trimmed oligosaccharide chains (Fig. 2,middle, B glycoform) unlike free, unassembled Vα11 proteins, which existed as both A and B glycoforms (Fig. 2,right-hand side). Newly synthesized Vα11 proteins bearing partially trimmed oligosaccharides (A glycoform) coprecipitated with calnexin in CD4+CD8+ thymocyte lysates, whereas those containing fully trimmed oligosaccharides (B glycoform) did not (Fig. 2, left-hand side). Thus, in agreement with previous studies on the processing of Vα11 proteins in splenic T lymphocytes (17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), these data show that Glc residues were removed from TCRα glycoproteins prior to their assembly with CD3ε chains in CD4+CD8+ thymocytes. Moreover, these data demonstrate that newly synthesized TCRα molecules containing partially trimmed oligosaccharides were associated with the molecular chaperone calnexin in CD4+CD8+ thymocytes. Interaction of calnexin with most newly synthesized glycoproteins is proposed to occur by a two-step process involving initial recognition of monoglucosylated glycans, followed by protein-protein interactions, which stabilize these associations (3Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recent studies show, however, that for certain glycoproteins, polypeptide contacts do not appear necessary for stable calnexin assembly (27Rodan A.R. Simmons J.E. Trombetta E.S. Helenius A. EMBO J. 1996; 15: 6921-6930Crossref PubMed Scopus (139) Google Scholar, 28Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). We reasoned therefore that TCRα proteins might precariously assemble with calnexin in CD4+CD8+ thymocytes via predominantly carbohydrate binding, such that TCRα polypeptide domains were particularly vulnerable to proteolytic degradation mechanisms. To investigate this issue, digitonin lysates of radiolabeled CD4+CD8+ thymocytes and splenic T cells were immunoprecipitated with anti-calnexin Ab; precipitates were either mock-treated or digested with Endo H, and association of TCRα proteins with calnexin before and after deglycosylation was compared. Calnexin interaction with TCRα proteins was unaffected by removal of oligosaccharides in CD4+CD8+ thymocytes and splenic T cells as similar amounts of TCRα molecules were associated with calnexin in mock and Endo H digested groups of both cell types (Fig. 3). These data demonstrate that TCRα proteins are stably associated with calnexin in CD4+CD8+ thymocytes and show that similar to splenic T cells, once formed, calnexin·TCRα protein complexes do not require oligosaccharide chains to maintain their association in CD4+CD8+ thymocytes. To investigate the quantitative assembly of newly synthesized TCRα proteins with lectin-like chaperones in CD4+CD8+ thymocytes and splenic T lymphocytes, cells were metabolically labeled with [35S]methionine for 30 min and digitonin lysates exhaustively immunoprecipitated with anti-calnexin Ab, anti-calreticulin Ab, and anti-TCRα Abs; sufficient Ab was used in these experiments to capture >95% of the respective antigens (data not shown). CD4+CD8+ thymocyte and splenic T lysates were immunoprecipitated with anti-TCRα Ab, anti-calnexin Ab, or anti-calreticulin Ab; precipitates were boiled in SDS to release bound material and TCRα proteins specifically recaptured using anti-TCRα mAb. As demonstrated, TCRα assembly with calnexin was quantiatively similar in CD4+CD8+thymocytes and splenic T cells (Fig. 4 A). These results were confirmed in sequential immunoprecipitation experiments in which lysates were precipitated with anti-calnexin Ab, followed by anti-TCRα mAb to capture remaining TCRα molecules (Fig. 4 B). In agreement with previous studies on calreticulin-TCRα association in other T cell types (13van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), much fewer TCRα proteins coprecipitated with calreticulin relative to calnexin in splenic T cells (Fig. 4 A), presumably because of the transient nature of calreticulin-TCRα interactions compared with calnexin-TCRα interactions (13van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Suprisingly, few, if any TCRα proteins were present in anti-calreticulin precipitates of CD4+CD8+ thymocyte lysates (Figs. 4 A), even upon prolonged exposure of autoradiographs; calreticulin expression was comparable in both cell types (data not shown). Taken together, these data show that calnexin association with newly synthesized TCRα proteins is quantitatively similar in CD4+CD8+ thymocytes and splenic T cells. In addition, these data contained the surprising finding that calreticulin association with TCRα proteins was specifically decreased in CD4+CD8+ thymocytes compared with splenic T cells. Recent studies in various nonlymphoid cell types expressing transfected TCRα gene products and murine BW thymoma cells have implicated proteasomes in the degradation of TCRα proteins (29Inoue S. Simoni R.D. J. Biol. Chem. 1992; 267: 9080-9086Abstract Full Text PDF PubMed Google Scholar, 30Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 31Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-836Crossref PubMed Scopus (202) Google Scholar). Because TCRα proteins synthesized in CD4+CD8+ thymocytes are significantly less stable than TCRα proteins expressed in other cell types, including T lymphocytes (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar), we wished to determine the involvement of proteasome activity in TCRα degradation in CD4+CD8+ thymocytes. For these studies we utilized ALLN, an inhibitor of various proteases including the proteasome and lactacystin, a highly specific proteasome inhibitor (32Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-836Crossref PubMed Scopus (202) Google Scholar,33Wada I. Ou W.J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar). In addition, we examined the effects of TPCK, a cysteine protease inhibitor (29Inoue S. Simoni R.D. J. Biol. Chem. 1992; 267: 9080-9086Abstract Full Text PDF PubMed Google Scholar) and diamide, a thiol-oxidizing agent previously reported to retard TCRα degradation in nonlymphoid cells (29Inoue S. Simoni R.D. J. Biol. Chem. 1992; 267: 9080-9086Abstract Full Text PDF PubMed Google Scholar) and to both induce and inhibit dissociation of calnexin with newly synthesized proteins (11Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 33Wada I. Ou W.J. Liu M.C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar). In agreement with previous studies on TCRα degradation in other cell types (29Inoue S. Simoni R.D. J. Biol. Chem. 1992; 267: 9080-9086Abstract Full Text PDF PubMed Google Scholar, 30Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 31Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-836Crossref PubMed Scopus (202) Google Scholar), the stability of unassembled TCRα proteins in CD4+CD8+ thymocytes was significantly enhanced by these reagents (Fig. 5, A and B). As shown in Fig. 6, inhibition of TCRα degradation in CD4+CD8+ thymocytes was correlated with increased stabilization of calnexin-TCRα protein complexes (Fig. 6). These data document that proteasome activity is important for the degradation of unassembled TCRα proteins in CD4+CD8+ thymocytes and show that impairment of proteasome activity results in protracted association of TCRα proteins with calnexin.Figure 6Inhibition of TCRα degradation is associated with increased stabilization of calnexin·TCRα protein complexes in CD4 + CD8 + thymocytes. CD4+CD8+ thymocytes were radiolabeled with [35S]methionine for 30 min, and chased in medium containing excess nonradioactive methionine and diamide (DIAM) (1 mm), TPCK (10 μg/ml), or ALLN (100 μg/ml) as indicated. Digitonin lysates were immunoprecipitated with anti-calnexin Ab; precipitates were boiled in SDS, and TCRα proteins were recaptured by precipitation with anti-TCRα mAb. The position of TCRα proteins is indicated. These data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Finally, we wished to examine the effects of the persistence of Glc residues onN-glycans on the assembly and degradation of TCRα proteins in CD4+CD8+ thymocytes. For these studies, CD4+CD8+ thymocytes were radiolabeled in the presence or absence of the glucosidase inhibitor castanospermine (cas), which blocks removal of Glc residues from N-glycan chains (34Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (348) Google Scholar). As expected, TCR glycoproteins synthesized in cas-treated CD4+CD8+ thymocytes migrated with decreased mobility compared with those made in untreated CD4+CD8+ thymocytes (Fig. 7 A), because of persistence of Glc residues on N-glycan chains. Most notably, these results demonstrate that TCRα proteins effectively assembled with CD3 components in cas-treated CD4+CD8+ thymocytes and that the stability of unassembled TCRα proteins was similar in media- and cas-treated CD4+CD8+ thymocytes (Fig. 7 A); these findings were confirmed by immunoprecipitation/release/recapture experiments (Fig. 7 B). These studies show that TCRα assembly with CD3 proteins is not precluded by the failure to remove Glc residues fromN-glycans on TCRα proteins and that degradation of unassembled TCRα proteins is not influenced by glucosidase activity in CD4+CD8+ thymocytes. ζ chain addition is the terminal stage of TCR assembly and depends on prior formation of αδε TCR intermediates (20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Because TCRα proteins are inherently unstable in CD4+CD8+ thymocytes, few, if any, αδε TCR intermediates or unassembled TCRα proteins exist within the ER that are available for assembly with newly translated ζ proteins (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Accordingly, the assembly of radiolabeled ζ chains with CD3 proteins approximates the rate of formation of complete TCR complexes in CD4+CD8+thymocytes (16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). As shown in Fig. 7 A, similar amounts of CD3-assembled ζ proteins were present in media- and cas-treated CD4+CD8+ thymocytes. Thus, we further conclude that persistence of Glc residues on TCRα proteins does not significantly affect the formation of complete TCR complexes in CD4+CD8+ thymocytes. Glc-containing high mannose oligosaccharides that escape the ER can be processed by endomannosidase enzymes localized in the intermediate ER/cis-Golgi compartment that cleave 1–3 Glc saccharides in association with a single mannose residue (2Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar, 34Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (348) Google Scholar), creating high mannose intermediates that are identical to those generated via conventional processing routes (2Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar); unlike ER glucosidase activity, endomannosidase action is insensitive to castanospermine treatment (35Moore S.E.H. Spiro R.G. J. Biol. Chem. 1992; 267: 8443-8451Abstract Full Text PDF PubMed Google Scholar). To determine whether CD3-assembled TCRα proteins were ultimately transported from the ER to the Golgi in cas-treated CD4+CD8+ thymocytes, radiolabeled cells were chased for 150 min. As shown in Fig. 7 A, CD3-associated TCRα proteins in cas-treated CD4+CD8+thymocytes acquired Endo H resistance during longer chase times (Fig. 7 A), indicative of their transport from the ER to at least the media Golgi compartment (2Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar, 26Tarentino A.L. Maley F. J. Biol. Chem. 1974; 249: 811-816Abstract Full Text PDF PubMed Google Scholar). Consistent with these findings, Endo H-resistant CD3δ,γ glycoproteins were also visible in anti-CD3 precipitates of chase groups of cas-treated CD4+CD8+ thymocytes (Fig. 7 A, see * in figure legend). Note that glucosidase activity was effectively inhibited throughout the course of these experiments as unassembled TCRβ proteins in cas-treated CD4+CD8+thymocytes showed increased mobility relative to media-treated CD4+CD8+ thymocytes during the entire chase period (data not shown). These data demonstrate that TCR complexes formed under conditions of cas-induced glucosidase blockade in CD4+CD8+ thymocytes effectively transit to at least the medial Golgi compartment. The current study has examined and compared the processing of TCRα proteins and their association with the lectin-like chaperones calnexin and calreticulin in immature CD4+CD8+thymocytes and mature splenic T cells. Our data demonstrate that TCRα glycoproteins are processed in a similar manner in both cell types, with Glc residues being removed from TCRα proteins before their assembly with CD3 components in CD4+CD8+thymocytes, as previously reported for splenic T cells. TCRα molecules were stably associated with calnexin in both cell types, but interestingly, calreticulin·TCRα associations were decreased in CD4+CD8+ thymocytes relative to splenic T cells. Finally, these studies show that TCRα proteins effectively assemble with CD3 subunits and egress from the ER to the Golgi complex under conditions where Glc residues persist on N-glycans and demonstrate that proteasome activity is important for TCRα degradation in CD4+CD8+ thymocytes. Because perturbation of Glc-trimming and calnexin assembly pathways markedly destabilizes TCRα proteins in other T cell types (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 17van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), it was hypothesized that inefficient Glc removal fromN-glycan chains and impaired calnexin assembly might represent the molecular basis for rapid degradation of TCRα proteins in CD4+CD8+ thymocytes (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar, 19van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The data in the current report effectively rule out this postulate, however, as these studies show that TCRα proteins synthesized in CD4+CD8+ thymocytes are effective substrates for Glc-trimming enzymes and associate with calnexin in a manner that is qualitatively and quantitatively similar to that which is observed in splenic T cells. Importantly, these studies establish that rapid degradation of TCRα proteins is not invariably correlated with the failure to associate with calnexin, suggesting that additional components in the ER quality control system (calreticulin?) operate to ensure the proper folding of nascent TCRα glycoproteins. Consistent with this concept, recent studies by Hebert et al. (12Hebert D.N. Zhang J.X. Chen W. Foellmer B. Helenius A. J. Cell Biol. 1997; 139: 613-623Crossref PubMed Scopus (221) Google Scholar) demonstrate that calnexin and calreticulin interact with distinct regions on newly synthesized influenza hemagglutinin molecules and associate with distinct hemagglutinin-folding intermediates. Calnexin and calreticulin have also been shown to associate with major histocompatibility complex class I proteins at different stages of their maturation and assembly (14van Leeuwen J.E.M. Kearse K.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13997-14001Crossref PubMed Scopus (76) Google Scholar, 36Sadasivan B. Lehner P.J. Ortmann B. Spies T. Cresswell P. Immunity. 1996; 5: 103-114Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Thus, it is reasonable to speculate that calnexin and calreticulin may perform specialized functions in the folding and assembly of TCRα proteins and that interaction with both chaperones is requisite for TCRα stability. Alternatively, it is possible that calreticulin association may intrinsically confer stability on TCRα proteins, an interaction that was shown to be specifically decreased in CD4+CD8+ thymocytes in this report. Data from previous studies on TCRα instability are concordant with all of these ideas as TCRα interaction with both calnexin and calreticulin chaperones is precluded under conditions of impaired glucosidase activity (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 19van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It remains to be determined, however, why calreticulin association with newly synthesized TCRα proteins is specifically decreased in immature CD4+CD8+thymocytes. Because TCRα proteins are inherently unstable in CD4+CD8+ thymocytes and their survival is unaffected by the persistence of Glc residues on N-glycan chains, we were able to significantly extend previous results onN-glycan processing and TCR assembly in other T cell types (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar) that were confounded by the differential stability of TCRα proteins under these conditions. Indeed, the data in the current report document that TCRα molecules effectively assemble with CD3 proteins and are transported to the Golgi complex when glucosidase activity is inhibited in CD4+CD8+ thymocytes. These results were hitherto unrealized because blockade of Glc removal from N-glycan chains dramatically destabilizes TCRα proteins in other T cell types (15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 16Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar, 18Kearse K.P. Roberts J.L Wiest D.L. Singer A. BioEssays. 1995; 17: 1049-1054Crossref PubMed Scopus (14) Google Scholar), making it difficult to determine the efficacy of TCR assembly under these conditions. The current data suggests, therefore, that although persistence of Glc residues on N-glycans severely limits the survival of newly synthesized TCRα proteins, TCRα molecules that survive under these conditions can effectively assemble into TCR complexes that are capable of ER to Golgi transport. Recent data from numerous laboratories indicate that misfolded proteins localized in the ER are degraded by cytoplasmic proteasomes, previously implicated only in the disposal of cytosolic proteins (37Kopito R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). Consistent with this idea, studies in nonlymphoid cells expressing a transfected TCRα gene product and murine BW thymoma cells suggest a model in which unassembled TCRα proteins are dislocated from the ER and degraded by cytosolic proteasomes (30Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 31Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-836Crossref PubMed Scopus (202) Google Scholar). In the current study we extend these findings to primary lymphocytes by showing that TCRα degradation in CD4+CD8+ thymocytes was markedly impaired by inhibitors of proteasome activity. Interestingly, inclusion of proteasome inhibitors stabilized calnexin·TCRα complexes in CD4+CD8+ thymocytes, suggesting that TCRα proteins may be degraded when associated with calnexin, or alternatively, that feedback mechanisms exist that regulate the dislocation of TCRα proteins in relation to proteasome activity (30Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Calnexin association is not requisite for disposal of unasssembled TCRα proteins in CD4+CD8+ thymocytes, however, as TCRα degradation was similar in the presence or absence of the glucosidase inhibitor, castanospermine, which effectively blocks the initial assembly of TCRα proteins with calnexin (Refs. 13van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and 15Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar; and data not shown). Finally, it is noteworthy to mention that instability of TCRα proteins and their interaction with lectin-like chaperones in CD4+CD8+ thymocytes shows several interesting parallels to recent observations on the biosynthesis of tyrosinase molecules in normal versus malignant melanocytes (38Halaban R. Cheng E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1987; 94: 6210-6215Crossref Scopus (232) Google Scholar). Analogous to the differential survival of nascent TCRα proteins in immature and mature T cells, the stability of tyrosinase proteins is significantly decreased in melanoma cells compared with normal melanocytes. Moreover, tyrosinase stably associates with both calnexin and calreticulin in normal melanocytes but solely with calnexin in malignant melanocytes (38Halaban R. Cheng E. Zhang Y. Moellmann G. Hanlon D. Michalak M. Setaluri V. Hebert D.N. Proc. Natl. Acad. Sci. U. S. A. 1987; 94: 6210-6215Crossref Scopus (232) Google Scholar). Thus, the differential stability of TCRα proteins in CD4+CD8+ thymocytes and splenic T cells represents the second example where rapid degradation of a newly synthesized protein is correlated with impaired calreticulin association but not calnexin association. Importantly, we would also note that similar to what has been reported regarding wild typeversus mutant proteins that contain defined singular amino acid changes, such as cystic fibrosis transmembrane conductor regulator molecules and α1-antitrypsin (39Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar, 40Thomas P.J. Qu B.H. Pedersen P.L. Trends Biol. Sci. 1995; 20: 456-459Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 41Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 42Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), calnexin assembly with newly synthesized TCRα proteins in immature and mature T cells does not distinguish between the dramatically different fates of TCRα proteins in these two cell types. Thus, undefined mechanisms must exist that regulate the assembly of TCRα proteins with CD3 components and TCRα survival in CD4+CD8+ thymocytes and splenic T cells. Further identification of quality control components necessary for the proper folding and stability of nascent TCRα proteins should provide important information regarding the regulation of TCRα stability in these cell types. We thank Drs. Mark Mannie, Tom McConnell, and Allan Weissman for critical reading of the manuscript and Dr. Stephen Hedrick for the gift of Vα11+ Tg mice.