Abstract
The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1.TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1.TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1.TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1.TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.
Highlights
Major histocompatibility complex (MHC)1 class I molecules are a complex of a heavy chain, a light chain (2-microglobulin), and a short peptide
In parallel sets of analyses, we observed a 2-fold reduction in the affinity calculated for TAP11⁄7TAP2-tapasin complexes (KD ϭ 50.4 Ϯ 5.05 nM; average of three experiments) compared with TAP11⁄7TAP2 complexes (KD ϭ 24.3 Ϯ 2.4 nM, average of two experiments in the present analyses; we previously reported room temperature peptide binding constants of 14.4 Ϯ 11 nM for TAP11⁄7TAP2 complexes in the absence of exogenous nucleotides (17) and 19.4 Ϯ 4.8 nM for TAP11⁄7TAP2 complexes in the presence of apyrase (6), which were derived from sets of analyses independent from the data described here)
We investigated the thermolability of the peptide binding site (Fig. 5) to determine whether it was accompanied by TAP11⁄7TAP2 dissociation and whether tapasin was still associated with the transporter associated with antigen processing (TAP) proteins upon incubation of the proteins at 34 °C
Summary
Baculoviruses for expression of, TAP1, TAP2, T1MT2C, T2MT1C, T1M, T2M, T1Ctr, T2Ctr, TAP1-eGFP, and Tapasin—Baculoviruses encoding wild type TAP1 and TAP2 constructs were obtained from the Tampe lab (16). The PCR products were ligated into pPCRscript (Stratagene), sequenced, and excised and ligated into the BamHI and BglII sites of the baculovirus transfer vector pAcUW51 (Pharmingen), respectively This vector was co-transfected with BaculoGold DNA (Pharmingen) into insect cells as described in the Pharmingen Baculovirus Expression Manual. Preparations of Microsomes, Immunoblotting Analyses, Peptide Translocation Assays, and Fluorescent Peptide-based Binding Assays— Cells were infected with appropriate baculoviruses (TAP1, TAP2 or TAP1, TAP2, tapasin) at multiplicity of infection values of [1– 80] depending on the desired protein expression levels. TAP and tapasin expression in the microsomes was verified by immunoblotting analyses of the microsome preparations (6) For this analysis, membranes were incubated in 15 ml of antibody buffer containing 50 l of 148.3 hybridoma supernatant (16), 10 l of 435.3 ascites fluid (20), and 7 l of anti-tapasin antiserum (13). Ratios of the relative intensities of TAP2 at 4 and 34 °C were quantified for the anti-TAP2 and anti-tapasinbased immunoprecipitations
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