Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

Abstract

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.

Figure 1

A2 and B51 alleles interact with transporter associated with antigen processing (TAP)1 and TAP2 expressed singly in T2 cells. T2 (a), T2rTAP1 (b) and T2rTAP2 (c) cells were metabolically radiolabelled and lysed in digitonin-containing buffers, followed by immunoprecipitation with antibodies recognizing human leucocyte antigen (HLA)-A, -B and -C, W6/32 (a), rat TAP1 (b) or rat TAP2 (c). Samples were analysed by two-dimensional electrophoresis. Only the portion of the gel containing the major histocompatibility complex (MHC) class I molecules is shown. The location of human tapasin is indicated by an arrowhead, and was confirmed by immunoprecipitation with anti-tapasin reagents and mass spectrometry (data not shown).

Figure 2

Multiple human leucocyte antigen (HLA) alleles interact with both transporter associated with antigen processing (TAP)1 and TAP2 subunits. T2rTAP1 and T2rTAP2 cells were infected with recombinant vaccinia viruses encoding HLA-B*2704, B*4402, B*3501 or A*2501 alleles. After metabolic radiolabelling and lysis in digitonin buffers, TAP1 and TAP2 were immunoprecipitated and samples analysed by two-dimensional electrophoresis. Only the portion of the gel containing the major histocompatibility complex (MHC) class I molecule is shown. Vaccinia (vacc)-encoded HLA alleles binding to TAP1 or TAP2 subunits are indicated by arrowheads. Immunoprecipitations of duplicate lysates were performed with W6/32 to control for possible differences in expression in the two cell lines (data not shown). Infections with HLA-negative viruses yielded results that were identical to the ‘no virus’ control.

Figure 3

Formation of major histocompatibility complex (MHC) class I/chaperone complexes by transporter associated with antigen processing (TAP) subunits. (a) T2rTAP1 cells were metabolically radiolabelled, lysed in digitonin and immunoprecipitated with antibodies to calreticulin (CRT), ERp57 or tapasin. Arrowheads indicate the position of chaperones, as determined by mass spectrometry and immunoprecipitation in Triton-X-100 lysis buffers (not shown). Only the portion of the gels containing MHC class I and chaperones are shown. The position of a contaminant identified as actin is indicated by the small letter ‘a’. (b) T2rTAP1 cells were metabolically radiolabelled, lysed in digitonin buffers and the lysate split in three portions. Lysates were immunoprecipitated with the monoclonal antibody W6/32 (first lane), or consecutively with anti-CRT or anti-ERp57 reagents, followed by anti-ERp57 or anti-CRT, respectively. Samples were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). (c) TAP1- and TAP2-associated A2 molecules are stabilized by peptide. T2rTAP1 and T2rTAP2 cells were metabolically radiolabelled, lysed in digitonin and split into three portions. After immunoprecipitation of TAP polypeptides, complexes were incubated with or without A2-binding peptide (LLDVPTAAV), dissociated from TAP and reimmunoprecipitated with W6/32, as described in the Materials and methods. Samples were analysed by SDS–PAGE.

Figure 4

Human leucocyte antigen (HLA)-A2 molecules dissociate from single transporter associated with antigen processing (TAP) subunits. (a) T2 and T2rTAP1 cells were lysed in digitonin and immunoprecipitated with antibodies to TAP1. Samples were analysed by two-dimensional electrophoresis and gels stained with Coomassie Blue. Anti-TAP1 antibody H- and L-chains are indicated by asterisks. Actin is shown as a small letter ‘a’. The identities of major histocompatibility complex (MHC) class I and chaperones were confirmed by mass spectrometry (except for β₂-microglobulin, which was confirmed by immunoprecipitation). TAP subunits do not resolve accurately during the isoelectric focusing step and do not appear on these gels. (b) T2rTAP1, T2rTAP2 and T2rTAP1+2 cells were metabolically radiolabelled and the samples either lysed immediately or chased for 1 hr and then lysed, followed by immunoprecipitation of TAPs. Only the major histocompatibility complex (MHC) class I region of the gel is shown. (c) Densitometric analysis of the TAP-associated A2 signal from (b). (d) T2rTAP1 cells were metabolically radiolabelled in the presence of protease inhibitors LLnL, lactacystin or control dimethylsulphoxide (DMSO). Cells were chased for 90 min, lysed in digitonin and TAP1 immunoprecipitated. Samples were analysed by two-dimensional electrophoresis. Only the region of the gels containing MHC class I heavy chain (and tapasin) spots are shown.

Figure 5

Single transporter associated with antigen processing (TAP) subunits bind ATP. (a) T2, T2rTAP1, T2rTAP2 and T1rTAP1+2 cells were lysed in Triton-X-100-containing buffer and incubated with ATP-agarose beads. Washed complexes were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting for TAP1 and TAP2. Control lysates are shown below each ATP-agarose gel. (b) The ATP-agarose isolation experiment from (a) was repeated with T2rTAP1 and T2rTAP2 cell lysates supplemented with or without 0·5 mm ATP. (c) Single TAP subunits were immunoprecipitated from T2rTAP1, T2rTAP2 or control T2 cells, and the immune complexes cross-linked to 8-azidoadenosine 5′[α-³²P]triphosphate. Sample were analysed by SDS–PAGE.

Figure 6

Chemical cross-linking reveals a transporter associated with antigen processing (TAP)2 homodimer. (a) Intact T2rTAP1, T2rTAP2 and T2rTAP1+2 cells were incubated with increasing concentrations of ethylene glycol bissuccinimidyl succinate (EGS). Cells were then lysed in Triton-X-100-containing buffer and analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting for TAP1 or TAP2. The arrow indicates the formation of a cross-linked TAP product. (b) T2rTAP2 and T2rTAP1+2 cells were lysed in digitonin (D) or Triton-X-100 (T) buffers prior to cross-linking with EGS. Samples were than analysed by SDS–PAGE and Western blotting for TAP2. An asterisk indicates the position of an additional, as yet unidentified, cross-linked product. (c) Digitonin lysates of T2rTAP2 and T2rTAP1+2 cells were incubated with or without 20 µm of TAP-binding peptide (TNKTVARYV) prior to EGS cross-linking. Samples were analysed by SDS–PAGE and Western blotting for TAP2.