Abstract
The transporter associated with antigen processing (TAP) comprises two structurally related subunits, TAP1 and TAP2, that form stable complexes in endoplasmic reticulum (ER) membranes. TAP complexes function in the translocation of peptides from the cytosol into the ER lumen for presentation by major histocompatibility complex class I molecules. Each TAP subunit contains an N-terminal membrane-spanning region with multiple membrane-spanning segments, and a C-terminal, cytosolic nucleotide binding region. To study the nature of the interactions occurring on the cytosolic face of TAP1/TAP2 complexes, we investigated quaternary associations mediated by two C-terminal fragments of human TAP1 (T1c, residues 452-748 and T1ctr, residues 472-748) and two C-terminal fragments of human TAP2 (T2c, residues 399-686 and T2ctr, residues 433-686). Each of these constructs contains the core nucleotide binding region as well as a long or short N-terminal extension. We show stable complex formation between T1c and T2c but not between T1ctr and T2ctr. The mechanistic implications of these results are discussed. We also show that each of the constructs except T1ctr interacts with wild type TAP1 and TAP2, indicating possibilities for homodimerization of TAP1 and TAP2, or of oligomerization of TAP1/TAP2 heterodimers on membranes.
Highlights
The TAP1 transporter is an essential component of the major histocompatibility complex class I antigen presentation pathway [1, 2]
To study the nature of the interactions occurring on the cytosolic face of TAP1/TAP2 complexes, we investigated quaternary associations mediated by two C-terminal fragments of human TAP1 (T1c, residues 452–748 and T1ctr, residues 472–748) and two C-terminal fragments of human TAP2 (T2c, residues 399 – 686 and T2ctr, residues 433– 686)
TAP1 and TAP2 form a complex in endoplasmic reticulum (ER) membranes, with the nucleotide binding domains (NBD) oriented in the cytosol [3]
Summary
Construction of Baculoviruses Encoding TAP1 and TAP2 NBD (T1c, T2c, T1cT2c, T1ctr, and T2ctr)—The cDNA for human TAP1 and TAP2 was obtained from the laboratory of Dr John Trowsdale and the following modifications introduced. The fusion constructs were individually subcloned into the BamHI or BglII sites of the baculovirus transfer vector pAcUW51 (PharMingen) to generate the viruses T1c, T2c, T1ctr, and T2ctr For this purpose, the corresponding recombinant transfer vectors and BaculoGold DNA (PharMingen) were co-transfected into insect cells as described in the PharMingen Baculovirus Expression Manual. After approximately 72 h, cells were lysed in 1 ml of ice-cold lysis buffer containing the protease inhibitor mixture, and the lysate was centrifuged at 40,000 ϫ g for 45 min. Samples were processed for immunoprecipitation as described above, separated on 10% polyacrylamide gels by SDS-PAGE, gels dried on a Bio-Rad model 483 slab dryer, and exposed directly to a PhosphorImager plate. Immunoprecipitated proteins were transferred from the gels to nitrocellulose membranes and analyzed by immunoblotting with anti-TAP1 and anti-TAP2 antibodies following protocols described above. A similar procedure was used to quantify the amounts of co-precipitating protein for each co-immunoprecipitation experiment shown in Figs. 3 and 5
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