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

The transporter associated with antigen processing (TAP) comprises two subu nits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is requ ired for peptide translocation across the endoplasmic reticulum membrane. T o understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MS R and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C. TAP2/T1MT2C, and T1MT2C/ T2MT1C complexes bind peptide with an affinity comparable to wild-type comp lexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observe d, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 a re required for binding peptide. However, neither NBD contains unique deter minants required for peptide binding. The NBD-switched complexes, T1MT2C/T2 MT1C. TAP1/T2MT1C, and TAP2/T1MT2C. all translocate peptides, but with prog ressively reduced efficiencies relative to the TAP1/TAP2 complex. These res ults indicate that both nucleotide binding sites are catalytically active a nd support an alternating catalytic sites model for the TAP transport cycle , similar to that proposed for P-glycoprotein. The enhanced translocation e fficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads . Preferential ATP interaction with TAP1, if occurring in vivo, might polar ize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP N BDs can mediate translocation indicate that distinct properties of the nucl eotide binding site per se are not essential for the TAP catalytic cycle.