Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution

Abstract

Previous studies of the yeast Saccharomyces cerevisiae indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these methods, we determined that zinc levels in the vacuole vary with zinc status and can rise to almost 100 mM zinc (i.e., 7 x 10(8) atoms of vacuolar zinc per cell). Moreover, this zinc can be mobilized effectively to supply the needs of as many as eight generations of progeny cells under zinc starvation conditions. While the Zrc1 and Cot1 zinc transporters are essential for zinc uptake into the vacuole under steady-state growth conditions, additional transporters help mediate zinc uptake into the vacuole during "zinc shock," when zinc-limited cells are resupplied with zinc. In addition, we found that other compartments of the cell do not provide significant stores of zinc. In particular, zinc accumulation in mitochondria is low and is homeostatically regulated independently of vacuolar zinc storage. Finally, we observed a strong correlation between zinc status and the levels of magnesium and phosphorus accumulated in cells. Our results implicate zinc as a major determinant of the ability of the cell to store these other important nutrients.

FIG. 1.

Effect of zinc status on accumulation and distribution of zinc. Wild-type (WT) and zrc1Δ cot1Δ mutant cells were grown in zinc-deficient CSD medium or in SD medium supplemented with the indicated concentrations of ZnCl₂. After 20 h, cells were harvested in log phase and assayed for (A) total cell-associated zinc, by AAS, and for (B) vacuolar zinc and (C) mitochondrial zinc, by ICP-MS analysis of isolated organelles. The data reported are the means of at least three independent cultures, and the error bars represent ±1 standard error. nd, not determined (zrc1Δ cot1Δ cells are not viable under those conditions of growth).

FIG. 2.

Analysis of element distribution in cells by EPXMA. Wild-type cells were grown in either (A) zinc-deficient CSD medium or (B) SD medium supplemented with 100 μM ZnCl₂. The cells were then cryopreserved, cryosectioned, and analyzed for element distribution by use of EPXMA. The STEM panels show scanning electron micrographs of 0.1-μm-thick sections of several yeast cells. The other panels show energy-dispersive X-ray maps of Ca, P, and Zn from the same sections. These maps are presented as false color images using the scale shown below the cell images. The arrows indicate the vacuole in one of the zinc-treated cells. Note that vacuoles are not apparent in some cells in the field because this compartment was not included in these particular sections of those cells. min, minimum; max, maximum.

FIG. 3.

Effects of the vacuolar zinc store on cell growth under zinc starvation conditions. Wild-type (WT) and zrc1Δ cot1Δ cells were grown overnight in SD medium supplemented with the indicated zinc concentrations. These cells were then washed free of extracellular zinc and inoculated into LZM with no added zinc, i.e., a medium with essentially no bioavailable zinc. (A) Cell densities of the cultures were monitored by measuring the OD₆₀₀ of the cultures after 28 h of incubation. The cell densities were then converted to the numbers of population doublings. Wild-type and mutant cells pregrown in SD medium with no added zinc and then inoculated into LZM plus 100 μM ZnCl₂ were used as a control for zinc-replete growth (+Zn control). (B) Total cellular zinc contents of the cells were measured by AAS after 28 h of growth. The data reported are the means of three independent cultures, and the error bars represent ±1 standard error. nd, not determined (zrc1Δ cot1Δ cells are not viable under those conditions of growth).

FIG. 4.

Effects of zinc shock on the accumulation and intracellular distribution of zinc. (A) Wild-type (WT) and zrc1Δ cot1Δ mutant cells were grown in zinc-limiting CSD medium overnight to induce zinc deficiency. The cells were then harvested, and their cell walls were removed to generate spheroplasts; this allowed the rapid isolation of organelles assayed as reported below. To induce zinc shock, 100 μM ZnCl₂ was added to the medium for a 10-min period. After this treatment, the cells were washed free of exogenous zinc and analyzed or incubated for an additional 10-min (10 + 10) or 20-min (10 + 20) chase period without zinc prior to harvesting. Cellular zinc levels were determined using AAS. In addition, vacuoles were isolated from wild-type cells (B), mitochondria were isolated from wild-type and zrc1Δ cot1Δ cells (C), and zinc levels were measured by ICP-MS. Vacuoles from zrc1Δ cot1Δ cells could not be obtained in this analysis due to organelle fragility; the cause of this fragility is unknown. The data reported are the means of three independent cultures, and the error bars represent ±1 standard error.

FIG. 5.

Element distribution in wild-type cells. Wild-type cells were grown in SD medium without additional zinc for 20 h and harvested in log phase. The cells were then cryopreserved and analyzed for element distribution by use of EPXMA. Energy-dispersive X-ray maps were analyzed, and concentrations of different elements (Ca, Cl, Fe, K, Mg, and P) in (A) whole cells and in (B to G) vacuoles (gray columns) and nonvacuolar cytoplasm (black columns) were determined. The means are reported, and the error bars indicate ±1 standard error. DW, dry weight.

FIG. 6.

Correlation between Mg and P contents in individual cells. Nonvacuolar cytoplasmic, vacuolar, and whole-cell levels of Mg and P, measured in individual cells grown in low (CSD medium), moderate (SD medium), and high (SD medium plus 100 μM ZnCl₂) zinc, are plotted. Wild-type values are indicated with circles, and zrc1Δ cot1Δ mutant values are indicated with “+” symbols. All values are in nmol/mg (dry weight).

FIG. 7.

Correlation between Mg, P, and K contents versus Zn content in individual cells. Vacuole and whole-cell levels of Mg, P, K, and Zn, measured in individual wild-type cells grown in high zinc (SD medium plus 100 μM ZnCl₂), are plotted. All values are in nmol/mg (dry weight).