Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11 168] we presented evidence for a novel Na+- and Cl--dependent Hf transport process in LR73/hu MDR 1 CHO transfectants that likely explains pH(i), volu me, and membrane potential changes in eukaryotic cells overexpressing the h u MDR 1 protein. To further explore this process, we have overexpressed hum an MDR 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 ta ndem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters t o create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confi rmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly , relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains ar e found to strongly depend on the ionic composition of the growth medium. A tomic absorption reveals significant differences in intracellular K+ for 9. 3/hu MDR I versus control yeast. Transport assays using [H-3]tetraphenylpho sphonium ([H-3]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pH(ex). ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Cen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast Hf AT Pase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of Hf gradients +/- vera pamil. Similar pharmacologic profiles are found for verapamil stimulation o f MDR 1 ATPase activity and Hf pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes Hf transpor t in some fashion and lowers membrane potential in yeast when Ki contribute s strongly to that potential. In the accompanying paper [Santai, C. T., Fri tz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined.