title: Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis.
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.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt/Main, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Herget
authors.forename: Nina
authors.initials: N
authors.lastname: Kreissig
authors.forename: Christian
authors.initials: C
authors.lastname: Kolbe
authors.forename: Christian
authors.initials: C
authors.lastname: Schölz
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D010455:Peptides; C477327:transporter associated with antigen processing (TAP); D000255:Adenosine Triphosphate