Molecular chaperons of SMF and Zn+2 metal ion transporters.
Noa Alumot (1), Adiel Cohen (1), Nathan Nelson (2)
(1) Biochemistry, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978, Israel; (2) Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv, University, Tel Aviv 69978, Israel
The mammalian and yeast metal ion transporters DCT1 and Smf1p were expressed in yeast and Xenopus oocytes. Sit-directed mutagenesis was utilized for the identification of the metal ions and proton binding sites. In DCT1 metal ions cause large proton slippages. In two DCT1 mutants this proton slip was nullified. The physiological significance of slips will be discussed. We also identified in yeast a novel family of genes named ZSP1, ZSP2, ZSP3, and ZSP4 for Zinc Sensitivity Phenotype, as well as a second family of genes STC1, STC2 and STC3 for Smf Transporters Chaperon. ZSP1 was identified as a suppressor of the Zn+2 sensitivity of the triple mutant smf1,2,3?. The null mutant zsp1? exhibited Zn+2 sensitivity and it may function as a vacuolar Zn+2 transporter. A mutation in a novel gene STC1 on the background of zsp1? suppressed the Zn+2 sensitivity and conferred EGTA sensitivity. STC1 has another two homologues in the yeast genome - STC2 and STC3. The EGTA sensitivity could not be overcome by overexpressing SMF1 or SMF2 but could be suppressed by a short version of Smf1p lacking the first 68 amino acids (Smf1p-s) or by overexpressing the rat homologue Dct1. We propose that Stc1p and Stc2p to be positive chaperons for Smf1p and Smf2p, responsible for their activation only in the late stages of the secretory pathway. The interaction between Smf1p and Stc2p may be mediated through the N-terminus of Smf1p.