Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Abstract

SummaryYcf1p is a member of the ATP-binding cassette transporter family of membrane 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 residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between 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 function relative to the wild-type protein.These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

Multiple Yap1p-binding Sites Mediate Induction of the Yeast Major Facilitator FLR1 Gene in Response to Drugs, Oxidants, and Alkylating Agents

“…MD was also included in the assay. We found that overexpression of (43). Similarly, overexpression of YAP1 in MRY13-1A caused a significant level of resistance to DEM that was reduced by the flr1⌬ deletion, demonstrating that YAP1 overexpression confers resistance to DEM and that FLR1 contributes to this resistance.…”

ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily

“…Examples include the antioxidant/oxidizing agents diamide and 1-chloro-2,4-dinitrobenzene (94,163). A potentially useful Ycf1p substrate that has yet to be fully exploited is monochlorobimane, which can be added to living yeast cells and visualized by fluorescence microscopy.…”

“…This (102,103). An unusual aspect of Ycf1p biology is that this transporter is posttranslationally processed to yield N-and C-terminal cleavage products that are stable and remain tightly associated with one another (102,163). Cleavage occurs within luminal loop 6 (L6) in a region representing an insertion that is specific to Ycf1p (Fig.…”

The Rho1 GTPase Acts Together With a Vacuolar Glutathione S-Conjugate Transporter to Protect Yeast Cells From Oxidative Stress

“…Does Tus1 activate Rho1 on the vacuolar membrane? Rho1 localizes to the plasma membrane and to other sites including bud tips, the mother-bud neck, and endomembranes (McCaffrey et al 1991;Drgonova et al 1996;Qadota et al 1996;Yoshida et al 2009), while Ycf1 localizes to the vacuolar membrane (Wemmie and Moye-Rowley 1997;Mason and Michaelis 2002). Tus1 localizes to the presumptive bud site in unbudded cells and to the mother-bud neck during cytokinesis (Yoshida et al 2006;Kono et al 2008), but has not been observed on the vacuolar membrane.…”