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J. Biol. Chem., Vol. 281, Issue 52, 39839-39851, December 29, 2006

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Catalytic Site Modifications of TAP1 and TAP2 and Their Functional Consequences^*

From the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620

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.

Received for publication, June 8, 2006 , and in revised form, October 3, 2006.

Note Added in Proof—Findings relevant to this study have recently been published (46).

^* This work was supported by National Institutes of Health Grant AI-44115 (to M. R.) and by an Investigator Award from the Cancer Research Institute (to M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: 5641 Medical Science Bldg. II, University of Michigan Medical School, Ann Arbor, MI 48109-0620. Tel.: 734-647-7752; Fax: 734-764-3562; E-mail: malinir{at}umich.edu.

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