Catalytic site modifications of TAP1 and TAP2 and their functional consequences

Abstract

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1·TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1·TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp668 in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln701 in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu632) follows the Walker B motif, and the switch region contains a histidine (TAP2 His661). We found that alterations at Glu632 and His661 of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp668 alone or in combination with TAP1 Gln701 had only small effects on TAP activity. Thus, the naturally occurring Asp668 and Gln701 alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.