PEPTIDE BINDING AND PHOTO-CROSS-LINKING TO DETERGENT-SOLUBILIZED AND TO RECONSTITUTED TRANSPORTER ASSOCIATED WITH ANTIGEN-PROCESSING (TAP)

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecu les by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally a ctive, heterologously expressed human TAP from microsomal membranes, T he efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions, The activity of t he solubilized TAP complex mas analyzed over time, using a non-radioac tive crosslinking assay,vith a photo-activateable peptide, in the pres ence or absence of external lipid, The detergent CHAPS mas found optim ally to retain activity and thus allowed us to reconstitute detergent- solubilized, active TAP into proteoliposomes. (C) 1997 Federation of E uropean Biochemical Societies.