High level expression, partial purification, and functional reconstitution of the human AE1 anion exchanger in Saccharomyces cerevisiae.
Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.
Sekler
I
I
Department of Biological Sciences, Stanford University, California 94305-5020, USA.
Kopito
R
R
Casey
J R
JR
D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D006801:Humans; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013431:Sulfates / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D004274:DNA, Recombinant; D013431:Sulfates