Detection of TAP family dimerizations by an in vivo assay in mammalian cells.
The transporter associated with antigen presentation (TAP) is an ATP-binding cassette (ABC) protein which transports peptides for presentation to the immune system. TAP is composed of two half transporters, TAP1 (ABCB2) and TAP2 (ABCB3), which heterodimerize to function. In humans, the TAP family consists of TAP1, TAP2, and TAPL (ABCB9). While the TAP1-TAP2 complex is well characterized, TAPL's dimerization state and function are unknown. To identify interactions within the human TAP family, we adapted the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to half ABC transporters. This assay has been shown to be suitable for the study of membrane-bound proteins in vivo [Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Science 283, 990-993]. With this method, in vivo TAP1-TAP2 heterodimerization was confirmed, no homodimerizations were detected with TAP1 or TAP2, and TAPL did not show any interaction with TAP1 or TAP2. However, we found strong evidence that TAPL forms homodimers. These results provide evidence of a novel homomeric TAPL interaction and demonstrate that the DHFR PCA will be of general utility in studies of half ABC transporter interactions in vivo.
Leveson-Gower
Dennis B
DB
British Columbia Cancer Research Centre, British Columbia Cancer Agency, University of British Columbia, Vancouver, V5Z 1L3 Canada.
Michnick
Stephen W
SW
Ling
Victor
V
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* / Q000502:physiology; D000818:Animals; D017951:Antigen Presentation / Q000235:genetics; D016466:CHO Cells; D002470:Cell Survival / Q000235:genetics; D002999:Clone Cells; D006224:Cricetinae; D019281:Dimerization; D005189:False Positive Reactions; D005434:Flow Cytometry; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D005810:Multigene Family; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C412573:ABCB9 protein, human; D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; C503300:Abcb9 protein, mouse; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C073119:TAP1 protein, human; C071692:TAP2 protein, human; D013762:Tetrahydrofolate Dehydrogenase