MUTATIONAL ANALYSIS OF THE SACCHAROMYCES-CEREVISIAE ATP-BINDING CASSETTE TRANSPORTER PROTEIN YCF1P

Ycf1p is a member of the ATP-binding cassette transporter family of me mbrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP), In this work, we have examined the functional significance of several of the conserved amino acid re sidues and the genetic requirements for Ycf1p subcellular localization . Biochemical fractionation experiments have established that Ycf1p, e xpressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps3 4 or end3 gene function, Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analo gous to those seen in the CFTR, An amino-terminal extension shared bet ween Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological functio n relative to the wild-type protein, These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the stu dy of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.