Detection of TAP Family Dimerizations by an in ViVo Assay in Mammalian Cells † Dennis B. Leveson-Gower, ‡ Stephen W. Michnick, § and Victor Ling* ,‡ British Columbia Cancer Research Centre, British Columbia Cancer Agency, UniVersity of British Columbia, VancouVer, V5Z 1L3 Canada, and UniVersite ´ de Montreal, Montre ´ al, H3C 3J7 Canada ReceiVed April 29, 2004; ReVised Manuscript ReceiVed September 3, 2004 ABSTRACT: 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. Perhaps the best characterized half ABC transporters are the ones which form the transporter associated with antigen presentation (TAP). 1 TAP translocates peptides from the cytoplasm into the lumen of the endoplasmic reticulum (ER), where they are assembled into major histocompatibility complex (MHC) class I molecules for presentation to the immune system (reviewed in ref 1). This transporter is a heterodimer consisting of TAP1 (ABCB2) and TAP2 (AB- CB3). Genetic evidence that TAP1 and TAP2 form het- erodimers comes from studies of human mutant cell lines which found that expression of both genes was needed for antigen processing (2-4). Co-immunoprecipitation studies indicate that TAP1 and TAP2 are found as a complex in the ER membrane (5, 6). This TAP heterodimer has been shown to function without any additional factor of the immune system (7, 8). A direct physical interaction has also been demonstrated between TAP1 and TAP2 when photoreactive peptide analogues labeled both TAP1 and TAP2, suggesting that the peptide-binding site of TAP is formed by amino acids from both TAP1 and TAP2 (9). Cross-linking experiments and gel filtration analysis suggest that TAP1 and TAP2 form a functional heterodimer with a stoichiometry of 1:1 (8, 10, 11). Transmission electron microscopy has since confirmed that TAP1 and TAP2 form a single heterodimeric complex (12). The third and final member of the TAP family is the TAP- like (TAPL, ABCB9) protein, which shares 38% and 40% amino acid sequence identity with TAP1 and TAP2, respec- tively. There is some debate over whether TAPL is localized to lysosomes or the endoplasmic reticulum, where TAP1 and TAP2 are localized (13, 14). High expression of TAPL was found in testis, and moderate expression was found in brain, spinal cord, and thyroid (13). Staining patterns seen with anti-TAPL antibody indicate that it is expressed in the Sertoli cells of the seminiferous tubules (13), which form part of the blood-testis barrier separating spermatogonia from spermatocytes and spermatids. Although its function is unknown, TAPL’s high degree of amino acid sequence identity with TAP1 and TAP2 suggests that it may be a peptide transporter and that it may dimerize with TAP1 or TAP2. While it is well-known that TAP1 forms a heterodimer with TAP2, there is also some evidence that TAP1 may form homodimers (15, 16). When rat TAP1 was introduced into murine small cell lung carcinoma cells, the cells gained the ability to be recognized by specific cytotoxic T-lymphocytes (CTLs) when infected with vesicular stomatitis virus (VSV) (15). Furthermore, the introduction of rat TAP1 caused VSV peptides to bind to putative lumenal ER proteins, suggesting † This work was supported by research grants from the Canadian Institutes of Health Research (MOP 42560) and the National Cancer Institute of Canada (NCI 11410). V.L. is a Distinguished Scholar of the Michael Smith Foundation for Health Research. * To whom correspondence should be addressed. Tel: 604-877-6151. Fax: 604-877-6150. E-mail: vling@bccancer.bc.ca. ‡ University of British Columbia. § Universite ´ de Montreal. 1 Abbreviations: ABCB2, ATP-binding cassette subfamily B trans- porter 2; ABCB3, ATP-binding cassette subfamily B transporter 3; ABCB9, ATP-binding cassette subfamily B transporter 9; bp, base pairs; CHO, Chinese hamster ovary; CMV, cyto-megalo virus; CTL, cytotoxic T-lymphocyte; DHFR PCA, dihydrofolate reductase protein-fragment complementation assay; ER, endoplasmic reticulum; FACS, fluorescence- activated cell sorting; FBS, fetal bovine serum; HT, hypoxanthine and thymidine; MHC, major histocompatibility complex; PBS, phosphate- buffered saline; PCR, polymerase chain reaction; RT, reverse transcrip- tion; TAP, transporter associated with antigen presentation; TAP1, transporter associated with antigen presentation 1, also known as ABCB2; TAP2, transporter associated with antigen presentation 2, also known as ABCB3; TAPL, TAP-like protein, also known as ABCB9; SDS, sodium dodecyl sulfate; UTR, untranslated region; VSV, vesicular stomatitis virus. 14257 Biochemistry 2004, 43, 14257-14264 10.1021/bi0491245 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/16/2004