The Zrc1 is involved in zinc transport system between vacuole and cytosol in Saccharomyces cerevisiae

The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously we found that the expression of ZRC1 was induced when the intracellular zinc level was decreased. Zrc1 has six putative transmembrane domains and we determined that a Zrc1-GFP fusion protein was localized to the vacuolar membrane. The steady state level of intracellular zinc in a zrc1Delta mutant cultured in the zinc-abundant medium was lower than that in wild type. No distinct difference was observed in the basal activity of glyoxalase I, which is a cytosolic enzyme requiring zinc for catalytic function and is used here as a marker for cytosolic zinc-availability, between wild type and zrc1Delta mutant, although the activity was decreased much greater extent in the zrc1Delta mutant if the cells were exposed to the metal-limited medium. Similarly, the basal expression level of ZRC1-lacZ reporter gene in zrc1Delta mutant was the same as that in wild type; however, the fold of induction of ZRC1-lacZ expression in zrc1Delta mutant under the zinc-limited conditions was higher than that in the wild type. Based on these results, we present a tentative model for the function of Zrc1 as a mechanism to maintain the zinc homeostasis in yeast.     

Vacuolar zinc transporter Zrc1 is required for detoxification of excess intracellular zinc in the human fungal pathogen <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Zinc is an important transition metal in all living organisms and is required for numerous biological processes. However, excess zinc can also be toxic to cells and cause cellular stress. In the model fungus Saccharomyces cerevisiae, a vacuolar zinc transporter, Zrc1, plays important roles in the storage and detoxification of excess intracellular zinc to protect the cell. In this study, we identified an ortholog of the S. cerevisiae ZRC1 gene in the human fungal pathogen Cryptococcus neoformans. Zrc1 was localized in the vacuolar membrane in C. neoformans, and a mutant lacking ZRC1 showed significant growth defects under high-zinc conditions. These results suggested a role for Zrc1 in zinc detoxification. However, contrary to our expectation, the expression of Zrc1 was induced in cells grown in zinc-limited conditions and decreased upon the addition of zinc. These expression patterns were similar to those of Zip1, the high-affinity zinc transporter in the plasma membrane of C. neoformans. Furthermore, we used the zrc1 mutant in a murine model of cryptococcosis to examine whether a mammalian host could inhibit the survival of C. neoformans using zinc toxicity. We found that the mutant showed no difference in virulence compared with the wildtype strain. This result suggests that Zrc1-mediated zinc detoxification is not required for the virulence of C. neoformans, and imply that zinc toxicity may not be an important aspect of the host immune response to the fungus.     

Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution

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.     

Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae

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.     

Zinc status and vacuolar zinc transporters control alkaline phosphatase accumulation and activity in Saccharomyces cerevisiae

Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo . We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn²⁺ binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.     

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.     

A cytosolic domain of the yeast Zrt1 zinc transporter is required for its post-translational inactivation in response to zinc and cadmium

Nutrient metals such as zinc are both essential to life and potentially toxic if overaccumulated by cells. Non-essential toxic metals like cadmium can enter cells through the uptake transporters responsible for nutrient metal acquisition. Therefore, in the face of ever changing extracellular metal levels, organisms tightly control their intracellular levels of nutrient metals and prevent accumulation of toxic metals. We show here that post-translational inactivation of the yeast Zrt1 zinc uptake transporter is important for zinc homeostasis. During the transition from zinc-limiting to zinc-replete growth conditions (i.e. zinc shock), the Zrt1 transporter is ubiquitinated, endocytosed, and subsequently degraded in the vacuole. To further understand this process at a molecular level, we mapped a region of Zrt1 required for ubiquitination and endocytosis in response to zinc to a domain located on the intracellular surface of the plasma membrane. This domain is a critical cis-acting component of the metal signaling pathway that controls Zrt1 protein trafficking. Using mutant alleles defective for metal-responsive inactivation, we also show that Zrt1 inactivation may be an important mechanism for preventing cadmium uptake and toxicity in zinc-limited cells.     

A single amino acid change in the yeast vacuolar metal transporters ZRC1 and COT1 alters their substrate specificity

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Deltaccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.     

Cytosolic Superoxide Dismutase (SOD1) Is Critical for Tolerating the Oxidative Stress of Zinc Deficiency in Yeast

Zinc deficiency causes oxidative stress in many organisms including the yeast Saccharomyces cerevisiae. Previous studies of this yeast indicated that the Tsa1 peroxiredoxin is required for optimal growth in low zinc because of its role in degrading H₂O₂. In this report, we assessed the importance of other antioxidant genes to zinc-limited growth. Our results indicated that the cytosolic superoxide dismutase Sod1 is also critical for growth under zinc-limiting conditions. We also found that Ccs1, the copper-delivering chaperone required for Sod1 activity is essential for optimal zinc-limited growth. To our knowledge, this is the first demonstration of the important roles these proteins play under this condition. It has been proposed previously that a loss of Sod1 activity due to inefficient metallation is one source of reactive oxygen species (ROS) under zinc-limiting conditions. Consistent with this hypothesis, we found that both the level and activity of Sod1 is diminished in zinc-deficient cells. However, under conditions in which Sod1 was overexpressed in zinc-limited cells and activity was restored, we observed no decrease in ROS levels. Thus, these data indicate that while Sod1 activity is critical for low zinc growth, diminished Sod1 activity is not a major source of the elevated ROS observed under these conditions.