Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

doi:10.1073/pnas.121180198

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

^*Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510; and ^†Division of Immunology, Department of Pathology, Wellcome Trust Centre for Molecular Mechanisms in Disease, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, United Kingdom

Communicated by D. Bernard Amos, Duke University Medical Center, Durham, NC (received for review February 27, 2001)

Abstract

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

Footnotes

↵ ‡ J.T.K. and P.J.L. contributed equally to this work.
↵ § To whom reprint requests should be addressed. E-mail: pjl30{at}cam.ac.uk or peter.cresswell{at}yale.edu.
Abbreviations:

ER,

endoplasmic reticulum;

ABC,

ATP-binding cassette;

NBD,

nucleotide binding domain;

IAA,

iodoacetamide;

TLCK,

N-tosyl-l-lysyl-chloromethyl ketone;

EGS,

ethylene glycol bis (succinimidyl succinate);

TAP,

transporter associated with antigen processing