Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6

The yeast a-factor transporter Ste6 is a member of the ABC transporter fami ly and is closely related to human MDR1. We constructed a set of 26 Ste6 mu tants using a random mutagenesis approach. Cell fractionation experiments d emonstrated that most of the mutants, with the notable exception of those w ith alterations in TM1, are transported to the plasma membrane, the presump tive site of action of Ste6. Trafficking, therefore, does not seem to be af fected in most of the mutants. To identify regions in Ste6 that interact wi th the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were iden tified in TM12 and upstream of TM6. Surprisingly, these mutations also supp ressed the Walker A mutation G397D, which should be defective in ATP-bindin g and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[ alpha-P-32]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste 6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.