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 short peptides from the cytosol to the endoplasmic reticulum, where peptides assemble with class I molecules of the major histocompatibility complex. TAP is comprised of two subunits, termed TAP1 and TAP2. We produced recombinant vaccinia viruses that direct synthesis of the TAP subunits, either individually or together. Virus-encoded TAP is rapidly and efficiently assembled (t1/2 of 5 min or less) by cells and does not spontaneously assemble in detergent extracts. By confocal immunofluorescence microscopy, TAP1 when expressed alone or with TAP2 is largely, if not exclusively, localized to the endoplasmic reticulum. Metabolic labeling with [2-3H]mannose demonstrates that TAP1 (but not TAP2) possesses Asn-linked oligosaccharides, but the lack of binding of [35S]methionine-labeled TAP to concanavalin A-agarose suggests that the glycosylated form represents a minor population of TAP1. The two subunits of the assembled complex present in detergent extracts photolabeled equally with 8-azido-[alpha-32P]ATP. Photolabeling of the two subunits was inhibited in parallel by various di- and trinucleotides, suggesting that their nucleotide binding sites function in a highly similar manner. Incubation of detergent extracts at 37 degrees C results in the rapid loss of TAP1 immunoreactivity, indicating either an unusual sensitivity to proteases or an irreversible conformation alteration.
Russ
G
G
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0440, USA.
Esquivel
F
F
Yewdell
J W
JW
Cresswell
P
P
Spies
T
T
Bennink
J R
JR
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003902:Detergents; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D008285:Major Histocompatibility Complex; D018613:Microscopy, Confocal; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics*
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D009711:Nucleotides; C073119:TAP1 protein, human; C071692:TAP2 protein, human