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 Ca 2؉ , Fe 2؉ , and Mn 2؉ . 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 is an essential nutrient but toxic to cells with overaccumulation. For this reason, intracellular zinc levels are tightly controlled. In the yeast Saccharomyces cerevisiae, the Zrc1 and Cot1 proteins have been implicated in the storage and detoxification of excess zinc in the vacuole. Surprisingly, transcription of ZRC1 is induced in zinc-limited cells by the zinc-responsive transcription factor Zap1. We show here that this increase in ZRC1 expression is a novel mechanism of zinc homeostasis and stress tolerance. Zinc-limited cells also express high levels of the plasma membrane zinc uptake transporters. As a consequence, when zinc-limited cells are resupplied with small amounts of zinc, large quantities quickly accumulate in the cell, a condition we refer to as "zinc shock." We show here that ZRC1 and its induction in zinc-limited cells are required for resistance to this zinc shock. Experiments using the zincresponsive fluorophore FuraZin-1 as an indicator of vacuolar zinc levels indicated that Zrc1 is required for the rapid transport of zinc into the vacuole during zinc shock. We also present evidence that cytosolic zinc rises to higher levels in cells unable to sequester this excess zinc. Thus, the increase in ZRC1 expression occurs prior to the zinc shock stress for which this induction is important. We propose that this "proactive" strategy of homeostatic regulation, such as we document here for ZRC1, may represent a common but largely unrecognized phenomenon.

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.

Proton and sulfate inhibition of the obligatory chloride-chloride exchange of human erythrocytes was measured at 0~ to determine their mechanism of reaction with the anion transporter. The proton and sulfate that are co-transported by this mechanism at higher temperatures behaved as nontransported inhibitors at 0~We analyzed the data in terms of four molecular mechanisms: (1) HSO~-addition to the transporter; (2) ordered addition with the proton first; (3) ordered addition with the sulfate first; (4) random addition to the transporter. The Dixon plots of 1/Mcl vs.[SO4] at different proton concentrations were not parallel. Thus protons and sulfate ions were not mutually exclusive inhibitors. The slope of these Dixon plots was independent of pH above 7.0, which indicates that sulfate could bind to the unprotonated carrier and excludes the first two mechanisms. Protons were inhibitors of chloride flux in the absence of sulfate, which indicates that protons could bind to the unloaded carrier and excludes mechanism 3. The KI for sulfate was 4.35 + 0.36 mM. The pK for the protonatable group was 5.03 + 0.02. The binding of either a proton or sulfate to the carrier decreased the KI of the other by ninefold. The only simple mechanism consistent with the data is a randomordered mechanism with more transporters loaded with a sulfate than loaded with a proton at the pH and sulfate concentrations of plasma.

Sulfate influx into human red blood cells was measured at 0 and 22 degrees C at several fixed external pH values between 3 and 10. These cells had normal internal pH and chloride concentrations so that sulfate influx was not limited by the efflux half-cycle reactions. The flux was a Michaelis-Menten function of sulfate concentration at each pH with K1/2SO4 = 4-10 mM. External protons activated influx 100-fold at a single site with a pK = 5.9 at 22 degrees C and 5.5 at 0 degrees C. This pK is similar to the value 5.99 +/- 0.3 for external proton binding to the sulfate-loaded transporter at 0 degrees C (J. Gen. Physiol. 79: 87-114, 1982). The flux was stilbene sensitive even in valinomycin-treated cells and was independent of membrane potential. This proton-activated influx appears to be proton-sulfate cotransport. At high pH there was a proton-independent flux that was membrane potential and stilbene sensitive. This proton-insensitive flux appears to be SO4(2-)/Cl- exchange or net sulfate influx. The sulfate influx over the entire pH range may be described in terms of an equation for the sum of the influxes through these two pathways on band 3.