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Physical and Functional Association of the Major Histocompatibility Complex Class I Heavy Chain 3 Domain with the Transporter Associated with Antigen Processing

Kimary Kulig^*, Dipankar Nandi, Igor Bacik, John J. Monaco^¶, and Stanislav Vukmanovic^*

From the ^* Michael Heidelberger Division of Immunology, the Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York 10016; the Department of Biochemistry, Indian Institute of Science, Bangalore, India 560012; Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892; and the ^¶ Department of Molecular Genetics, Howard Hughes Medical Institute, University of Cincinnati, Cincinnati, Ohio 45267

CD8⁺ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain 3 domain. Substitution of a 15 amino acid portion of the H-2D^b 3 domain (aa 219-233) with the analogous MHC class II (H-2IA^d) β2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26°C in the presence of excess peptide and β2-microglobulin. Mutant H-2D^b (D^b219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the 3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

¹Abbreviations used in this paper: β2m, β2-microglobulin; ER, endoplasmic reticulum; ES, E3/19K leader/signal sequence; NEPHGE, nonequilibrium pH-gradient gel electrophoresis; NP, nucleoprotein; RP10, RPMI 1640/10% FCS; TAP, transporter associated with antigen processing; VV, vaccinia virus; wt, wild type.

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