Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.
We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.
Groves
J D
JD
Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
Falson
P
P
le Maire
M
M
Tanner
M J
MJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism; D006801:Humans; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D003513:Cycloheximide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D005690:Galactose