Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins.
The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.
Lapinski
Philip Edward
PE
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
Raghuraman
Gayatri
G
Raghavan
Malini
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera
D001386:Azides; D002352:Carrier Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C021622:8-azidoadenosine diphosphate