Fig. 7. Photolabeling of wild-type and mutant MRP1 proteins with either [α-³²P]-8-N₃ATP or [γ-³²P]-8-N₃ATP.

A. Walker A and B mutants mainly affect nucleotide binding. Membrane vesicles prepared in Figure 5 were used to do the photolabeling experiments. Photolabeling experiments were carried out in a 10 μl of solution containing 10 μg membrane vesicle proteins (the amount of MRP1 protein determined in Figure 5A was adjusted to a similar amount by adding varying amount of membrane vesicles prepared from parental BHK cells), 10 μM [α-³²P]-8-N₃ATP (α) or 10 μM [γ-³²P]-8-N₃ATP (γ) and 800 μM vanadate at 37 °C for 2 minutes. The reaction mixture was subjected to UV-irradiation (+) or without UV-irradiation (-) on ice for 2 minutes, separated by SDS-PAGE (7%) and electroblotted to a nitrocellulose membrane. Molecular weight markers (kDa) are indicated on the left. MRP1 on the right of the gel indicates the ³²P-nucleotide labeled MRP1 proteins. B. S685A-mutated MRP1 mainly affects ATP binding at the mutated NBD1, but not the ATP hydrolysis at the un-mutated NBD2. S685A or S685T mutation was introduced into the pDual/N-half/C-half and expressed in Sf21 insect cells [24]. The membrane vesicles prepared from the viral particle infected Sf21 cells were used to do photolabeling as described in panel A. NH and CH on the right indicate the ³²P-labeled NBD1-containing N-half and NBD2-containing C-half. All the experiments were repeated three times and the counts in each band were determined by Instant Imager [20].