Purification and reconstitution of the antigen transport complex TAP: a prerequisite for determination of peptide stoichiometry and ATP hydrolysis.
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.
Herget
Meike
M
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt/Main, Germany.
Kreissig
Nina
N
Kolbe
Christian
C
Schölz
Christian
C
Tampé
Robert
R
Abele
Rupert
R
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
D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D010455:Peptides; C477327:transporter associated with antigen processing (TAP); D000255:Adenosine Triphosphate