Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1996 Aug 1;184(2):337–348. doi: 10.1084/jem.184.2.337

MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains

PMCID: PMC2192707  PMID: 8760787

Abstract

Newly assembled heavy chain-beta 2m heterodimers of class I histocompatibility molecules associate with the endoplasmic reticulum (ER) peptide transporter, TAP, and subsequently dissociate from TAP in parallel with their transport from the ER to the Golgi apparatus. It appears that TAP-associated class I molecules are waiting to bind appropriate peptides before they dissociate from TAP and leave the ER since binding of high affinity peptides to class I molecules in vitro leads to dissociation of TAP-class I complexes. In further support of this notion, we report that limiting peptide supply through inhibition of proteasome activities prolongs the association of mouse class I molecules with TAP and concomitantly slows their transport to the Golgi apparatus. By using a series of deletion mutants and hybrid class I molecules we demonstrate that the extracellular domains of class I molecules are sufficient for their peptide-regulated interaction with TAP. Furthermore, based on the inability of an alpha 3 domain-specific mAb to recognize TAP-class I complexes and the fact that a point mutant of the Dd molecule at residue 222 is unable to bind to TAP, it is likely that a major site of interaction with TAP resides in the membrane-proximal region of the heavy chain alpha 3 domain. Finally, we examined the relationship between the interaction of mouse heavy chain- beta 2m heterodimers with TAP and with the resident ER chaperone, calnexin. Most heterodimers that bound to TAP were found to associate simultaneously with calnexin. Upon delivery of peptide to class I molecules in permeabilized cells, dissociation from TAP was observed but the interaction with calnexin was largely maintained. Therefore, both TAP and calnexin may participate in the ER retention of peptide- deficient class I molecules. However, since release from calnexin occurs after dissociation from TAP, it appears that calnexin ultimately determines if a class I molecule is to be exported from the ER.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abastado J. P., Jaulin C., Schutze M. P., Langlade-Demoyen P., Plata F., Ozato K., Kourilsky P. Fine mapping of epitopes by intradomain Kd/Dd recombinants. J Exp Med. 1987 Aug 1;166(2):327–340. doi: 10.1084/jem.166.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ajitkumar P., Geier S. S., Kesari K. V., Borriello F., Nakagawa M., Bluestone J. A., Saper M. A., Wiley D. C., Nathenson S. G. Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor. Cell. 1988 Jul 1;54(1):47–56. doi: 10.1016/0092-8674(88)90178-x. [DOI] [PubMed] [Google Scholar]
  3. Allen H., Fraser J., Flyer D., Calvin S., Flavell R. Beta 2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7447–7451. doi: 10.1073/pnas.83.19.7447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allen H., Wraith D., Pala P., Askonas B., Flavell R. A. Domain interactions of H-2 class I antigens alter cytotoxic T-cell recognition sites. Nature. 1984 May 17;309(5965):279–281. doi: 10.1038/309279a0. [DOI] [PubMed] [Google Scholar]
  5. Androlewicz M. J., Ortmann B., van Endert P. M., Spies T., Cresswell P. Characteristics of peptide and major histocompatibility complex class I/beta 2-microglobulin binding to the transporters associated with antigen processing (TAP1 and TAP2). Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12716–12720. doi: 10.1073/pnas.91.26.12716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bluestone J. A., Jameson S., Miller S., Dick R., 2nd Peptide-induced conformational changes in class I heavy chains alter major histocompatibility complex recognition. J Exp Med. 1992 Dec 1;176(6):1757–1761. doi: 10.1084/jem.176.6.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carreno B. M., Schreiber K. L., McKean D. J., Stroynowski I., Hansen T. H. Aglycosylated and phosphatidylinositol-anchored MHC class I molecules are associated with calnexin. Evidence implicating the class I-connecting peptide segment in calnexin association. J Immunol. 1995 May 15;154(10):5173–5180. [PubMed] [Google Scholar]
  8. Carreno B. M., Solheim J. C., Harris M., Stroynowski I., Connolly J. M., Hansen T. H. TAP associates with a unique class I conformation, whereas calnexin associates with multiple class I forms in mouse and man. J Immunol. 1995 Nov 15;155(10):4726–4733. [PubMed] [Google Scholar]
  9. Catipović B., Talluri G., Oh J., Wei T., Su X. M., Johansen T. E., Edidin M., Schneck J. P. Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface. J Exp Med. 1994 Nov 1;180(5):1753–1761. doi: 10.1084/jem.180.5.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Connolly J. M., Hansen T. H., Ingold A. L., Potter T. A. Recognition by CD8 on cytotoxic T lymphocytes is ablated by several substitutions in the class I alpha 3 domain: CD8 and the T-cell receptor recognize the same class I molecule. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2137–2141. doi: 10.1073/pnas.87.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Corr M., Boyd L. F., Padlan E. A., Margulies D. H. H-2Dd exploits a four residue peptide binding motif. J Exp Med. 1993 Dec 1;178(6):1877–1892. doi: 10.1084/jem.178.6.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Degen E., Cohen-Doyle M. F., Williams D. B. Efficient dissociation of the p88 chaperone from major histocompatibility complex class I molecules requires both beta 2-microglobulin and peptide. J Exp Med. 1992 Jun 1;175(6):1653–1661. doi: 10.1084/jem.175.6.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Degen E., Williams D. B. Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibility molecules. J Cell Biol. 1991 Mar;112(6):1099–1115. doi: 10.1083/jcb.112.6.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Früh K., Ahn K., Djaballah H., Sempé P., van Endert P. M., Tampé R., Peterson P. A., Yang Y. A viral inhibitor of peptide transporters for antigen presentation. Nature. 1995 Jun 1;375(6530):415–418. doi: 10.1038/375415a0. [DOI] [PubMed] [Google Scholar]
  15. Grandea A. G., 3rd, Androlewicz M. J., Athwal R. S., Geraghty D. E., Spies T. Dependence of peptide binding by MHC class I molecules on their interaction with TAP. Science. 1995 Oct 6;270(5233):105–108. doi: 10.1126/science.270.5233.105. [DOI] [PubMed] [Google Scholar]
  16. Heemels M. T., Ploegh H. Generation, translocation, and presentation of MHC class I-restricted peptides. Annu Rev Biochem. 1995;64:463–491. doi: 10.1146/annurev.bi.64.070195.002335. [DOI] [PubMed] [Google Scholar]
  17. Howard J. C. Supply and transport of peptides presented by class I MHC molecules. Curr Opin Immunol. 1995 Feb;7(1):69–76. doi: 10.1016/0952-7915(95)80031-x. [DOI] [PubMed] [Google Scholar]
  18. Jackson M. R., Cohen-Doyle M. F., Peterson P. A., Williams D. B. Regulation of MHC class I transport by the molecular chaperone, calnexin (p88, IP90). Science. 1994 Jan 21;263(5145):384–387. doi: 10.1126/science.8278813. [DOI] [PubMed] [Google Scholar]
  19. Joyce S., Tabaczewski P., Angeletti R. H., Nathenson S. G., Stroynowski I. A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides. J Exp Med. 1994 Feb 1;179(2):579–588. doi: 10.1084/jem.179.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kleijmeer M. J., Kelly A., Geuze H. J., Slot J. W., Townsend A., Trowsdale J. Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi. Nature. 1992 May 28;357(6376):342–344. doi: 10.1038/357342a0. [DOI] [PubMed] [Google Scholar]
  21. Little A. M., Nössner E., Parham P. Dissociation of beta 2-microglobulin from HLA class I heavy chains correlates with acquisition of epitopes in the cytoplasmic tail. J Immunol. 1995 May 15;154(10):5205–5215. [PubMed] [Google Scholar]
  22. Machold R. P., Andrée S., Van Kaer L., Ljunggren H. G., Ploegh H. L. Peptide influences the folding and intracellular transport of free major histocompatibility complex class I heavy chains. J Exp Med. 1995 Mar 1;181(3):1111–1122. doi: 10.1084/jem.181.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Madden D. R. The three-dimensional structure of peptide-MHC complexes. Annu Rev Immunol. 1995;13:587–622. doi: 10.1146/annurev.iy.13.040195.003103. [DOI] [PubMed] [Google Scholar]
  24. Margolese L., Waneck G. L., Suzuki C. K., Degen E., Flavell R. A., Williams D. B. Identification of the region on the class I histocompatibility molecule that interacts with the molecular chaperone, p88 (calnexin, IP90). J Biol Chem. 1993 Aug 25;268(24):17959–17966. [PubMed] [Google Scholar]
  25. McCluskey J., Boyd L., Foo M., Forman J., Margulies D. H., Bluestone J. A. Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants. J Immunol. 1986 Dec 15;137(12):3881–3890. [PubMed] [Google Scholar]
  26. Momburg F., Neefjes J. J., Hämmerling G. J. Peptide selection by MHC-encoded TAP transporters. Curr Opin Immunol. 1994 Feb;6(1):32–37. doi: 10.1016/0952-7915(94)90030-2. [DOI] [PubMed] [Google Scholar]
  27. Neefjes J., Gottfried E., Roelse J., Grommé M., Obst R., Hämmerling G. J., Momburg F. Analysis of the fine specificity of rat, mouse and human TAP peptide transporters. Eur J Immunol. 1995 Apr;25(4):1133–1136. doi: 10.1002/eji.1830250444. [DOI] [PubMed] [Google Scholar]
  28. Nössner E., Parham P. Species-specific differences in chaperone interaction of human and mouse major histocompatibility complex class I molecules. J Exp Med. 1995 Jan 1;181(1):327–337. doi: 10.1084/jem.181.1.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ortmann B., Androlewicz M. J., Cresswell P. MHC class I/beta 2-microglobulin complexes associate with TAP transporters before peptide binding. Nature. 1994 Apr 28;368(6474):864–867. doi: 10.1038/368864a0. [DOI] [PubMed] [Google Scholar]
  30. Rajagopalan S., Brenner M. B. Calnexin retains unassembled major histocompatibility complex class I free heavy chains in the endoplasmic reticulum. J Exp Med. 1994 Jul 1;180(1):407–412. doi: 10.1084/jem.180.1.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ribaudo R. K., Margulies D. H. Independent and synergistic effects of disulfide bond formation, beta 2-microglobulin, and peptides on class I MHC folding and assembly in an in vitro translation system. J Immunol. 1992 Nov 1;149(9):2935–2944. [PubMed] [Google Scholar]
  32. Rock K. L., Gramm C., Rothstein L., Clark K., Stein R., Dick L., Hwang D., Goldberg A. L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell. 1994 Sep 9;78(5):761–771. doi: 10.1016/s0092-8674(94)90462-6. [DOI] [PubMed] [Google Scholar]
  33. Rohren E. M., McCormick D. J., Pease L. R. Peptide-induced conformational changes in class I molecules. Direct detection by flow cytometry. J Immunol. 1994 Jun 1;152(11):5337–5343. [PubMed] [Google Scholar]
  34. Rötzschke O., Falk K., Stevanović S., Grahovac B., Soloski M. J., Jung G., Rammensee H. G. Qa-2 molecules are peptide receptors of higher stringency than ordinary class I molecules. Nature. 1993 Feb 18;361(6413):642–644. doi: 10.1038/361642a0. [DOI] [PubMed] [Google Scholar]
  35. Salter R. D., Benjamin R. J., Wesley P. K., Buxton S. E., Garrett T. P., Clayberger C., Krensky A. M., Norment A. M., Littman D. R., Parham P. A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2. Nature. 1990 May 3;345(6270):41–46. doi: 10.1038/345041a0. [DOI] [PubMed] [Google Scholar]
  36. Smith M. H., Parker J. M., Hodges R. S., Barber B. H. The preparation and characterization of anti-peptide heteroantisera recognizing subregions of the intracytoplasmic domain of class I H-2 antigens. Mol Immunol. 1986 Oct;23(10):1077–1092. doi: 10.1016/0161-5890(86)90006-4. [DOI] [PubMed] [Google Scholar]
  37. Solheim J. C., Carreno B. M., Myers N. B., Lee D. R., Hansen T. H. Peptide-induced rescue of serologic epitopes on class I MHC molecules. J Immunol. 1995 Feb 1;154(3):1188–1197. [PubMed] [Google Scholar]
  38. Sugita M., Brenner M. B. An unstable beta 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide. J Exp Med. 1994 Dec 1;180(6):2163–2171. doi: 10.1084/jem.180.6.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Suh W. K., Cohen-Doyle M. F., Fruh K., Wang K., Peterson P. A., Williams D. B. Interaction of MHC class I molecules with the transporter associated with antigen processing. Science. 1994 May 27;264(5163):1322–1326. doi: 10.1126/science.8191286. [DOI] [PubMed] [Google Scholar]
  40. Tector M., Salter R. D. Calnexin influences folding of human class I histocompatibility proteins but not their assembly with beta 2-microglobulin. J Biol Chem. 1995 Aug 18;270(33):19638–19642. doi: 10.1074/jbc.270.33.19638. [DOI] [PubMed] [Google Scholar]
  41. Thor G., Sepulveda H., Chada S., Dutton R. W. Monoclonal antibody that distinguishes between a phosphorylated, beta 2-microglobulin-associated, and a free, nonphosphorylated, chain of MHC class I. J Immunol. 1993 Jul 1;151(1):211–224. [PubMed] [Google Scholar]
  42. Townsend A., Ohlén C., Bastin J., Ljunggren H. G., Foster L., Kärre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature. 1989 Aug 10;340(6233):443–448. doi: 10.1038/340443a0. [DOI] [PubMed] [Google Scholar]
  43. Vassilakos A., Cohen-Doyle M. F., Peterson P. A., Jackson M. R., Williams D. B. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. EMBO J. 1996 Apr 1;15(7):1495–1506. [PMC free article] [PubMed] [Google Scholar]
  44. Ware F. E., Vassilakos A., Peterson P. A., Jackson M. R., Lehrman M. A., Williams D. B. The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J Biol Chem. 1995 Mar 3;270(9):4697–4704. doi: 10.1074/jbc.270.9.4697. [DOI] [PubMed] [Google Scholar]
  45. Williams D. B., Barber B. H., Flavell R. A., Allen H. Role of beta 2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules. J Immunol. 1989 Apr 15;142(8):2796–2806. [PubMed] [Google Scholar]
  46. Williams D. B. The Merck Frosst Award Lecture 1994/La conference Merck Frosst 1994. Calnexin: a molecular chaperone with a taste for carbohydrate. Biochem Cell Biol. 1995 Mar-Apr;73(3-4):123–132. doi: 10.1139/o95-015. [DOI] [PubMed] [Google Scholar]
  47. Yokoyama K., Geier S. S., Uehara H., Nathenson S. G. Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity. Biochemistry. 1985 Jun 4;24(12):3002–3006. doi: 10.1021/bi00333a029. [DOI] [PubMed] [Google Scholar]
  48. Zhang Q., Tector M., Salter R. D. Calnexin recognizes carbohydrate and protein determinants of class I major histocompatibility complex molecules. J Biol Chem. 1995 Feb 24;270(8):3944–3948. doi: 10.1074/jbc.270.8.3944. [DOI] [PubMed] [Google Scholar]
  49. van Endert P. M., Riganelli D., Greco G., Fleischhauer K., Sidney J., Sette A., Bach J. F. The peptide-binding motif for the human transporter associated with antigen processing. J Exp Med. 1995 Dec 1;182(6):1883–1895. doi: 10.1084/jem.182.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES