Abstract
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-β-D-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 nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg2+, and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.
