Biochemical characterization of transporter associated with antigen processing (TAP)-like (ABCB9) expressed in insect cells.
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-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 Mg(2+), and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.
Ohara
Tomomi
T
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
Ohashi-Kobayashi
Ayako
A
Maeda
Masatomo
M
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D019281:Dimerization; D004330:Drosophila; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility
C412573:ABCB9 protein, human; D018528:ATP-Binding Cassette Transporters; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate