INTERACTIONS & MECHANISMS OF FUNCTION OF THE TAP COMPLEX

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The transporters associated with antigen processing (TAP) are crucial components of the class I major histocompatibility complex (MHC) antigen presentation pathway. TAP is a complex of two subunits, TAP1 and TAP2, that functions to transport peptides from the cytosol to the endoplasmic reticulum (ER) for class I MHC antigen presentation. TAP1 and TAP2 both comprise a transmembrane domain and an ATP binding domain (NBD). The studies proposed here will result in a better understanding of TAP function and insights into molecular mechanisms by which the immune system combats viruses and cancers. In the first of the proposed studies, the interactions of ATP with human TAP subunits will be analyzed and structural changes that are induced as a consequence of ATP binding will be probed using proteolysis, circular dichroism and surface plasmon resonance-based assays. In particular, experiments are proposed to determine whether nucleotide binding alters the stability of a complex that we observe between the NBD of TAP1 and TAP2. In the second of the proposed studies, the question of whether ATP binding and hydrolysis by both TAP1 and TAP2 subunits is required for peptide translocation by TAP is addressed, by the generation and characterization of mutants that are predicted to alter the ATPase activities of one of the TAP subunits. The basis for these studies is the observation of natural mutations in human and rodent TAP2 sequences at sites that are highly conserved and that have been shown to be crucial for ATPase activity and transport function of other members of the ATP binding cassette (ABC) family of transmembrane transporters (of which TAP is a member). In the last of proposed studies, the interactions of the TAP complex with the human class I MHC molecule HLA -B27 and the newly discovered TAP-associated protein, tapasin will be examined. Studies in other laboratories have indicated that newly synthesized class I MHC molecules form complexes with TAP in the ER, and that this interaction is bridged or stabilized by tapasin. By expressing different combinations of the proteins TAP1 TAP2, tapasin, and the class I MHC heavy and light chains in insect cells, the molecular nature of the TAP/tapasin/class I MHC complexes will be elucidated, and insights will be obtained into whether other unknown components are required for these interactions, as well as for class I MHC antigen presentation. Finally, analysis of TAP interactions with HLA-B27 that are proposed will provide insight into the molecular mechanisms that underlie the association of B27 with autoimmune arthritic diseases.