CLONING AND FUNCTIONAL-CHARACTERIZATION OF A SUBUNIT OF THE TRANSPORTER ASSOCIATED WITH ANTIGEN-PROCESSING

The transporter associated with antigen processing (TAP) is essential for the transport of antigenic peptides across the membrane of the end oplasmic reticulum, In addition, TAP interacts with major histocompati bility complex class I heavy chain (HC)/beta 2-microglobulin (beta 2-m ) dimers. We have cloned a cDNA encoding a TAP1/2-associated protein ( TAP-A) corresponding in size and biochemical properties to tapasin, wh ich was recently suggested to be involved in class I-TAP interaction ( Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. & Cresswell, P. ( 1996) Immunity 5, 103-114), The cDNA encodes a 448-residue-long ORF, i ncluding a signal peptide, The protein is predicted to be a type I mem brane glycoprotein with a cytoplasmic tail containing a double-lysine motif (-KKKAE-COOH) known to maintain membrane proteins in the endopla smic reticulum, Immunoprecipitation with anti-TAP1 or anti-TAP-A antis era demonstrated a consistent and stoichiometric association of TAP-A with TAP1/2, Class I HC and beta 2-m also were coprecipitated with the se antisera, indicating the presence of a pentameric complex, In pulse -chase experiments, class I HC/beta 2-m rapidly dissociated from TAP1/ 2-TAP-A, We propose that TAP is a trimeric complex consisting of TAP1, TAP2, and TAP-A that interacts transiently with class I HC/beta 2-m. In peptide-binding assays using cross-linkable peptides and intact mic rosomes, TAP-A bound peptides only in the presence of ATP whereas bind ing of peptides to TAP1/2 was ATP-independent, This suggests a direct role of TAP-A in peptide loading onto class I HC/beta 2-m dimer.