Characteristics of peptide and major histocompatibility complex class I/beta 2-microglobulin binding to the transporters associated with antigen processing (TAP1 and TAP2).

Abstract

The transporter proteins associated with antigen processing (TAP proteins) transport antigenic peptides across the endoplasmic reticulum membrane where they can assemble with newly synthesized maijor histocompatibility complex (MHC) class I/p2-microglobulin (j32m) dimers. We have shown previously that TAP possesses a peptide-recognition site with broad specificity and that MHC class I/p2m dimers physically associate with TAP. Here, we further characterize the nature of the peptide-binding site on TAP, and the site of interaction of TAP with MHC class I/fi2m dimers. TAP photoaffinity labeling experiments revealed that both TAPi and TAP2 are photolabeled by two distinct photopeptide analogues, suggesting that elements of both TAPN and TAP2 compose the peptide-recognition site. TAP photolabeling analysis on transfectant cell lines that express TAPi and TAP2 both individually and together revealed that efficient formation of the peptide-binding site occurs only when TAPN and TAP2 are coexpressed, which correlates with the finding that peptide translocation via TAP occurs only in the presence of both TAPN and TAP2. These data strongly support the notion that TAP functions as a heterodimer. MHC class I/132m dimers were shown to associate with individual TAP1 chains but were not detectable with individual TAP2 chains. This result suggests that the site of interaction for MHC class I/A2m dimers with TAP is on TAP1.Traffic ATPases or ABC transporters (for ATP-binding cassette) constitute a large family of related molecules that are responsible for translocating a variety of compounds across membranes (for review, see ref. 1). Examples include the oligopeptide (OppABCDF) and hemolysin (HlyB) transporters in bacteria (2, 3), the STE6 (sterile 6) a-factor transporter in yeast (4), the eukaryotic P-glycoprotein associated with multidrug resistance (5, 6), and the cystic fibrosis transmembrane conductance regulator (7). Although many such transporters have been identified in prokaryotes and eukaryotes, limited information is available on the actual mechanism of translocation. Members of the ABC transporter family possess a characteristic structure consisting of two hydrophobic domains (each with six putative membrane-spanning segments) and two hydrophilic ATP-binding domains. The transporter proteins associated with antigen processing (TAP proteins) are recently identified members of the traffic ATPases (8-10). TAP molecules transport antigenic peptides across the endoplasmic reticulum (ER) membrane (11-13), an essential step in the major histocompatibility complex (MHC) class I antigen-presentation pathway. Through the action of TAP, peptides generated in the cytosol gain entry into the ER, where they can associate with MHC class I/02-microglobulin (f32m) heterodimers. TAP molecules are made up of two distinct polypeptides, termed TAP1 and 3!AP2, each containing single hydrophobic and ATP-binding domains. Normal peptide loading and class I transport can be reconstituted in mutant cells that lack both TAPl-and TAP2-...

Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP)

“…Studies with photoreactive peptides showed that both TAP subunits contribute to peptide binding (41). The peptide-binding pocket was localized in the cytosolic loops between predicted TM8 and TM9 of TAP1, TM7 and TM8 of TAP2, and a stretch of 15 amino acids C-terminal of TM10 of TAP1 and TM9 of TAP2 (42,43).…”

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

“…Further cross-linking experiments with radiolabeled peptides suggested that regions of the MSRs of TAP1 and TAP2, just N terminal to the NBD, form the peptide binding site (6). Neither TAP1 alone nor TAP2 alone is capable of binding peptides (4). The role of the NBD and nucleotides in peptide binding is controversial.…”

“…By contrast to peptide binding, it is well established that peptide translocation by TAP complexes is strictly ATPdependent (4,9). The two NBDs power the transport of peptides via the hydrolysis of ATP.…”

“…The structural organization of the TAP1͞TAP2 complex [two nucleotide binding domains (NBDs) and two membrane-spanning regions (MSRs)] is characteristic of the ATP binding cassette family of transmembrane transporters (3). Early studies showed that TAP complexes contained a binding site for peptides and that the peptide binding site comprised elements of both TAP1 and TAP2 (4,5). Further cross-linking experiments with radiolabeled peptides suggested that regions of the MSRs of TAP1 and TAP2, just N terminal to the NBD, form the peptide binding site (6).…”

Reconstitution of peptide‐binding activity by TAP in proteoliposomes

“…reactive peptide KB 11-HSAB was used to photolabel TAP as previously described [8]. 125 I-KB11-HSAB (200 nM) was added to the dishes, and incubation was carried out in the dark for 30 min on ice.…”