Biochemical characterization of transporter associated with antigen processing (TAP)-like (ABCB9) expressed in insect cells

摘要

The ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL) tagged with a histidine cluster was overexpressed, amounting to as much as 1-2% of total membrane proteins in Drosophila cell line S2. TAPL was effectively solubilized from membranes by Triton X-100, NP-40 and n-dodecyl-beta-o-maltoside. Solubilized TAPL bound ATP-agarose and adenosine 5'-diphosphate (ADP)agarose but not adenosine 5'-monophosphate (AMP)-agarose. The binding was competed for by excess free ATP, ADP, guanosine 5'-triphosphate (GTP) and dATP but not by AMP. Pyrimidine nucleotides such as uridine 5'-triphosphate (UTP) and cytidine 5'-triphosphate (CTP) were less effective competitors, suggesting that purine (2+) nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg, and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.