UBC Theses and Dissertations
Characterization of interactions in the TAP family of half ABC transporters Leveson-Gower, Dennis Brian
The transporter associated with antigen processing (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 require heterodimerization for function. In humans, the TAP gene family consists of TAP 1, TAP2, and TAPL (ABCB9). While the TAP1-TAP2 complex is well-characterized, the dimerization state and function of TAPL are unknown. To identify interactions within the human TAP family, I adapted the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to study the human genes coding for the half ABC transporters. This assay has been used for the study of membrane-bound proteins in vivo (Remy, I., Wilson, I. A., and Michnick, S. W. (1999) "Science" 283(5404), 990-3). 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, I 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. By using an insect-cell microsomal transport assay, I found the first direct evidence that TAPL can transport peptides. Two classical TAP substrates, RRYQNSTEL and RYWANATRST were transported by TAPL. Kinetic characterization of the transport of RYWANATRST indicated that TAPL follows Michaelis-Menton kinetics with an apparent K[sub m] of 3.2 ± 0.4 nM.
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