Evidence for Altered Ion Transport in Saccharomyces cerevisiae Overexpressing Human MDR 1 Protein

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• 作者：Friederike Fritz ; Ellen M. Howard ; Mary M. Hoffman ; and Paul D. Roepe
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：March 30, 1999
• 年：1999
• 卷：38
• 期：13
• 页码：4214 - 4226
• 全文大小：146K
• 年卷期：v.38,no.13(March 30, 1999)
• ISSN：1520-4995

文摘

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] wepresented evidence for a novel Na⁺- and Cl^--dependent H⁺ transport process in LR73/hu MDR 1 CHOtransfectants that likely explains pH_i, volume, and membrane potential changes in eukaryotic cellsoverexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed humanMDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K.,and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandemarray of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vectorpFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasmamembranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance andgrowth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growthmedium. Atomic absorption reveals significant differences in intracellular K⁺ for 9.3/hu MDR 1 versuscontrol yeast. Transport assays using [³H]tetraphenylphosphonium ([³H]TPP⁺) reveal perturbations inmembrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pH_ex. ATPase activityof purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectantsconstructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared.MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeastH⁺ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and controlstrains were analyzed for formation of H⁺ gradients ± verapamil. Similar pharmacologic profiles arefound for verapamil stimulation of MDR 1 ATPase activity and H⁺ pumping in 9.3/hu MDR 1 ISOV. Insum, these experiments strongly support the notion that hu MDR 1 catalyzes H⁺ transport in some fashionand lowers membrane potential in yeast when K⁺ contributes strongly to that potential. In the accompanyingpaper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects ofion gradients on H⁺ transport by hu MDR 1 are examined.