The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature-sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non-permissive temperature (37 C), was more sensitive to Zn than its parental GDI1 strain at 32 C (a moderately non-permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport-specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1-related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn-specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 C and 37 C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn.

A CPx-ATPase (named Bxa1) is induced in the cyanobacterium Oscillatoria brevis upon exposure to multiple heavy metal ions. The function of the bxa1 gene was examined by heterologous expression in both Saccharomyces cerevisiae (yeast) and Escherichia coli. Expression of bxa1 in E. coli caused Ag, Cd and Zn tolerance, but in yeast became sensitive to those metals. To reveal the role of the N-terminal His-rich domain (first 35 amino acids) of Bxa1, we constructed E. coli and yeast transformants carrying the bxa1 (Δ35bxa1). The E. coli transformant with Δ35bxa1 was sensitive to heavy metals. On the other hand, the yeast Δ35bxa1 transformant increased heavy-metal tolerance than bxa1 transformant. Fluorescence microscopy suggested that the two fusion proteins Bxa1::mGFP and Δ35Bxa1::mGFP are mainly localized in yeast endoplasmic reticulum (ER). These results imply that the function of Bxa1 was lost by the N-terminus deletion in both E. coli and yeast transformants. This is the first report that the His-rich domain in O. brevis Bxa1 is essential not only to monovalent (Ag+ and Cu+) but also to divalent (Cd2+ and Zn2+) heavy metal tolerance. Moreover, we clarified the toxicity mechanism against Cd using yeast transformants.