Expression and characterization of the anion transporter homologue YNL275w in 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 sequence similarity to the family of mammalian Cl-/HCO3- anion exchangers and Na+/HCO3- cotransporters. This yeast protein also has a very similar hydropathy profile to the mammalian HCO3- transporters, indicating a similar membrane topology and structure. A V5 epitope and His6-tagged version of Ynl275wp was expressed in yeast and was localized to the plasma membrane by subcellular fractionation and immunofluorescence labeling. The protein was purified by nickel affinity chromatography and was found not to be N-glycosylated. The protein's mobility on SDS-PAGE gels was not altered by treatment with N-glycanase F, alpha-mannosidase, or by mutation of each of the five consensus 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-Gel), and this binding could be competed by certain anions (HCO3-, Cl-, NO3-, and I-) but not by others (SO4(2-) and PO4(3-)). These results suggest that the yeast gene YNL275w encodes a nonglycosylated anion transport protein, localized to the plasma membrane.
Zhao
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Canadian Institutes of Health Research Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Reithmeier
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D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D027963:Chloride-Bicarbonate Antiporters; D002846:Chromatography, Affinity; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D005810:Multigene Family; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D027982:Sodium-Bicarbonate Symporters; D013104:Spheroplasts / Q000737:chemistry / Q000378:metabolism
D027321:Anion Transport Proteins; D000838:Anions; D017920:Antiporters; D001639:Bicarbonates; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D005656:Fungal Proteins; C434364:PMA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D027982:Sodium-Bicarbonate Symporters; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases