Expression and characterization of the anion transporter homologue YNL275win Saccharomyces cerevisiae

A search of the yeast Saccharomyces cerevisiae genome has revealed an open reading frame, YNL275w, which encodes a 576-amino acid protein that shows s equence similarity to the family of mammalian Cl-/HCO3- anion exchangers an d Na+/HCO3- cotransporters. This yeast protein also has a very similar hydr opathy profile to the mammalian HCO3- transporters, indicating a similar me mbrane topology and structure. A V5 epitope and His6-tagged version of Ynl2 75wp was expressed in yeast and was localized to the plasma membrane by sub cellular fractionation and immunofluorescence labeling. The protein was pur ified by nickel affinity chromatography and was found not to be N-glycosyla ted. The protein's mobility on SDS-PAGE gels was not altered by treatment w ith N-glycanase F, alpha -mannosidase, or by mutation of each of the five c onsensus N-glycosylation sites. The protein did not bind to concanavalin A by lectin blotting or lectin affinity chromatography. The expressed protein bound specifically to a stilbene disulfonate inhibitor resin (SITS-Affi-Ge l), and this binding could be competed by certain anions (HCO3-,Cl-,NO3-,an d I-) but not by others (SO42- and PO43-). These results suggest that the y east gene YNL275w encodes a nonglycosylated anion transport protein, locali zed to the plasma membrane.