Abstract
The yeast vacuole plays an important role in zinc homeostasis by storing zinc for later use under deficient conditions, sequestering excess zinc for its detoxification, and buffering rapid changes in intracellular zinc levels. The mechanisms involved in vacuolar zinc sequestration are only poorly characterized. Here we describe the properties of zinc transport systems in yeast vacuolar membrane vesicles. The major zinc transport activities in these vesicles were ATP-dependent, requiring a H+ gradient generated by the V-ATPase for function. One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters. These data are consistent with the proposed role of Zrc1 as a vacuolar zinc transporter. Zrc1-independent activity was also observed that was not dependent on the closely related vacuolar Cot1 protein. Both Zrc1-dependent and independent activities showed a high specificity for Zn(2+) over other physiologically relevant substrates such as Ca2+, Fe2+, and Mn2+. Moreover, these systems had high affinities for zinc with apparent K(m) values in the 100-200 nm range. These results provide biochemical insight into the important role of Zrc1 and related proteins in eukaryotic zinc homeostasis.
Highlights
Studies of the yeast Saccharomyces cerevisiae have uncovered many aspects of zinc homeostasis in this eukaryotic organism
One system we identified was dependent on the ZRC1 gene, which encodes a member of the cation diffusion facilitator family of metal transporters
Zinc Accumulation by Isolated Yeast Vacuole Vesicles—Many genetic studies have implicated the importance of vacuolar zinc transport in zinc homeostasis
Summary
The zinc sensitivity of yeast is significantly increased by the zrc mutation, such mutant strains are still tolerant to millimolar concentrations of zinc, which are unlikely to be encountered in the natural environment. Our recent studies suggest that an important physiological role of Zrc is to tolerate the rapid influx of zinc that results from exposing zinc-deficient yeast to zinc-replete conditions (zinc shock).. Okorokov et al [25] were the first to assay zinc transport activity in yeast vacuolar vesicles. These investigators characterized a transport system with an apparent Km of 55–170 M. White and Gadd [26] noted zinc transport activity in vacuole vesicles, but this activity was not further described Because of these considerations, we initiated this study of the biochemical properties of zinc transport activities in vacuolar vesicles. Using genetic manipulations in combination with our biochemical assays, we investigated the contribution of the CDF family proteins and the V-ATPase to vacuolar zinc transport
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