ASSEMBLY, INTRACELLULAR-LOCALIZATION, AND NUCLEOTIDE-BINDING PROPERTIES OF THE HUMAN PEPTIDE TRANSPORTERS TAP1 AND TAP2 EXPRESSED BY RECOMBINANT VACCINIA VIRUSES

The transporter associated with antigen processing (TAP) transports sh ort peptides from the cytosol to the endoplasmic reticulum, where pept ides assemble with class I molecules of the major histocompatibility c omplex, TAP is comprised of two subunits, termed TAP1 and TAP2, We pro duced recombinant vaccinia viruses that direct synthesis of the TAP su bunits, either individually or together, Virus-encoded TAP is rapidly and efficiently assembled (t(1/2) of 5 min or less) by cells and does not spontaneously assemble in detergent extracts, By confocal immunofl uorescence microscopy, TAP1 when expressed alone or with TAP2 is large ly, if not exclusively, localized to the endoplasmic reticulum, Metabo lic labeling with [2-H-3]mannose demonstrates that TAP1 (but not TAP2) possesses Asn-Linked oligosaccharides, but the lack of binding of [S- 35]methionine-labeled TAP to concanavalin A-agarose suggests that the glycosylated form represents a minor population of TAP1, The two subun its of the assembled complex present in detergent extracts photolabele d equally with 8-azido-[alpha-P-32]ATP. Photolabeling of the two subun its was inhibited in parallel by various di- and trinucleotides, sugge sting that their nucleotide binding sites function in a highly similar manner, Incubation of detergent extracts at 37 degrees C results in t he rapid loss of TAP1 immunoreactivity, indicating either an unusual s ensitivity to proteases or an irreversible conformation alteration.