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J Biol Chem. 2009 Dec 4;284(49):33740-9. doi: 10.1074/jbc.M109.047779. Epub 2009 Oct 6.

Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis.

Author information

  • 1Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt/Main, Germany.

Abstract

The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.

PMID:
19808685
[PubMed - indexed for MEDLINE]
PMCID:
PMC2797143
Free PMC Article

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