GOLGI LOCALIZATION AND FUNCTIONALLY IMPORTANT DOMAINS IN THE NH2 AND COOH TERMINUS OF THE YEAST CLC PUTATIVE CHLORIDE CHANNEL GEF1P

GEF1 encodes the single CLC putative chloride channel in yeast. Its di sruption leads to a defect in iron metabolism (Greene, J. R., Brown, N . H., DiDomenico, B. J., Haplan, J., and Eide, D. (1993) Mol. Gen. Gen et. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuo lar acidification. We now show that gef1 cells indeed grow less well a t less acidic pH. However, no defect in vacuolar acidification is appa rent from quinacrine staining, and Gef1p co-localizes with Mnt1p in th e medial Golgi, Thus, Gef1p may be important in determining Gels pH. S ystematic alanine scanning of the amino and the carboxyl terminus reve aled several regions essential for Gef1p localization and function. On e sequence (FVTID) in the amino terminus conforms to a class of sortin g signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identifi ed a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localizatio n The second CBS domain can be transplanted to the amino terminus with out loss of function, but could not be replaced by the corresponding d omain of the homologous mammalian channel CIC-2.