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 yeas t Saccharomyces cerevisiae under the control of the constitutive promo ter and transcriptional terminator of the yeast phosphoglycerate kinas e 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 r eticulum. AE1 protein was solubilized from yeast membranes with lysoph osphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation af finity chromatography, Size-exclusion chromatography in the presence o f octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stoke s radius of 77 Angstrom, consistent with a dimeric oligomeric state, B inding of partially purified yeast expressed AE1 to acetamido-4'-isoth iocyanostilbene-2,2'-disulfonate resin was competitive with the transp ortable substrate chloride but not the nontransported anion citrate, s uggesting that the structure of the anion binding site is preserved, T he specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authent ic AE1 purified from erythrocyte membranes, These data show that this expression system has the capacity to produce functional mammalian pla sma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.