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.
Highlights
The transporter associated with antigen processing (TAP)5 has a key role in the adaptive immune system of higher vertebrates
1.0 a To determine the efficiency in solubilization and recovery of TAP, the amount of TAP in crude membranes was compared with TAP after solubilization and purification by densitometry of immunoblots using monoclonal ␣-TAP1 (148.3) and ␣-TAP2 (435.3) antibodies. b The molar ratio of TAP1 and TAP2 in crude membranes was taken as 1
The activity of solubilized TAP could be checked in every step and lead to important results: (i) Peptide binding triggers ATP hydrolysis of TAP in the solubilized state
Summary
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. We evolved in a detailed screen a solubilization, purification, and reconstitution procedure for heterologously expressed TAP With this we addressed the peptide-binding stoichiometry and basal ATP hydrolysis of TAP
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