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. IMAGES:

我们通过可诱导型GAL10-CYC1启动子调控，在蛋白酶缺陷型酿酒酵母（Saccharomyces cerevisiae）菌株中表达人类红细胞阴离子转运蛋白（带3蛋白；AE1）的52kDa整合膜结构域（B3mem）。对转化酵母细胞的总蛋白进行免疫印迹检测，结果证实B3mem多肽在经半乳糖诱导后迅速过表达。通过简易转运实验检测完整酵母细胞中二苯乙烯二磺酸盐可抑制的氯离子内流，可证实功能性阴离子转运蛋白的细胞表面表达。B3mem多肽在细胞内的回收与降解半衰期约为1~3小时，这使得指数生长期培养物中该蛋白的表达达到稳态水平。我们的实验数据表明，任意时刻仅有总B3mem的5%~10%在细胞表面处于功能活性状态，且该阴离子转运蛋白的过表达不会干扰细胞生长与存活。本研究是为数不多的在酵母细胞质膜中功能性表达质膜转运蛋白的报道之一，且据我们所知，这是首项在cDNA转化细胞的质膜中实现红细胞带3蛋白介导的阴离子转运的研究。我们所描述的细胞表面表达系统，将为未来通过定点诱变研究带3蛋白的功能特性提供一种简便手段。图像：