The yeast mutant vps5Delta affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity

Abstract

Growth of the yeast vacuolar protein-sorting mutant vps5Delta affected in the endosome-to-Golgi retromer complex was more sensitive to Mg2+-limiting conditions than was the growth of the wild-type (WT) strain. This sensitivity was enhanced at acidic pH. The vps5Delta strain was also sensitive to Al3+, known to inhibit Mg2+ uptake in yeast cells. In contrast, it was found to be resistant to Ni2+ and Co2+, two cytotoxic analogs of Mg2+. Resistance to Ni2+ did not seem to result from the alteration of plasma-membrane transport properties because mutant and WT cells displayed similar Ni2+ uptake. After plasma-membrane permeabilization, intracellular Ni2+ uptake in vps5Delta cells was 3-fold higher than in WT cells, which is consistent with the implication of the vacuole in the observed phenotypes. In reconstituted vacuolar vesicles prepared from vps5Delta, the rates of H+ exchange with Ni2+, Co2+, and Mg2+ were increased (relative to WT) by 170%, 130%, and 50%, respectively. The rates of H+ exchange with Ca2+, Cd2+, and K+ were similar in both strains, as were alpha-mannosidase and H+-ATPase activities, and SDS/PAGE patterns of vacuolar proteins. Among 14 other vacuolar protein-sorting mutants tested, only the 8 mutants affected in the recycling of trans-Golgi network membrane proteins shared the same Ni2+ resistance phenotype as vps5Delta. It is proposed that a trans-Golgi network Mg2+/H+ exchanger, mislocalized to vps5Delta vacuole, could be responsible for the phenotypes observed in vivo and in vitro.

Figure 1

Growth phenotypes of vps5Δ and WT. (A) pH-dependent phenotypes in Mg²⁺-limiting media. Spot assays were recorded after 5-days (200 μM Mg²⁺, Mg²⁺-nonlimiting condition) or 10-days of growth (other assays, Mg²⁺-limiting conditions) on an agarose/YNB minimal medium at the indicated pH and Mg²⁺ concentrations. (B) Tolerance to cytotoxic heavy metals. Spot assays were recorded after 6-days of growth on an agar/YNB minimal medium (pH 4.3) containing 0.1 mM Mg²⁺ (nonlimiting), and the following concentrations of the indicated metals: 1 mM Al³⁺, 0.5 mM Co²⁺, 0.25 mM Ni²⁺, and 0.25 mM Cd²⁺. C, control plate.

Figure 2

⁶³Ni²⁺ uptake by vps5Δ and WT cells. (A) Control cells. (B) Cells with permeabilized plasma membrane. Values for WT (●) and vps5Δ (○) are presented as mean ± SE (n = 3). The uptake is expressed as mmol of Ni²⁺ per mg of dry weight.

Figure 3

Biochemical properties of tonoplast isolated from vps5Δ and WT. (A) Isolation yield, hydrolytic activity of V-ATPase and α-mannosidase expressed in mg of protein per g of fresh weight, μmol of ATP hydrolyzed per hr per mg of protein, and nmol of nitrophenol per min per mg of protein, respectively. These activities were not significantly different between the two strains (Student's t tests, data not shown). (B) In SDS/PAGE patterns of tonoplast proteins, 62 bands were detected in both patterns with image analysis software. (C) Western blot analysis of vacuole membrane (Vac) and subcellular P15, P150, and S150 fractions from WT and vps5Δ strains. For a given antibody, equal amounts of fraction proteins were loaded. Monoclonal antibodies were directed against membrane markers of the tonoplast (Vph1p), PVC (Pep12p), endoplasmic reticulum (Dpm1p), mitochondria (CoxIIIp), and TGN (Vps10p).

Figure 4

Activity of Ni²⁺/H⁺ exchange in reconstituted tonoplast vesicles from vps5Δ and WT. Ammonium-containing vesicles were diluted 200-fold in the assay buffer free of NH, causing vesicle acid-loading and instant quenching of the permeant pH probe ACMA. Fluorescence recovery after the addition of 0.5 mM Ni²⁺ to reconstituted tonoplast vesicles, not observed on control liposomes, ascertained the facilitated exchange activity. The final addition of NH (15 mM) to the outside allowed the total dissipation of the pH gradient. Solid and dotted lines represent the mean ACMA fluorescence in arbitrary units (au) ± SE (n = 5), respectively.

Figure 5

Selectivity of cation/proton exchange across reconstituted tonoplast vesicles from vps5Δ or WT. Cation concentration was 0.5 mM, except for Cd²⁺, which was 0.2 mM. Exchange rates (V_H⁺) are presented as mean V_H⁺ ± SE (n = 3), except for Mg²⁺ (n = 5). Exchange rates were significantly different between vps5Δ and WT for Mg²⁺, Co²⁺, and Ni²⁺, but not for Cd²⁺, Ca²⁺, and K⁺ (Student's t tests, data not shown).

Figure 6

Mg²⁺/H⁺ exchange rate of reconstituted tonoplast vesicles from vps5Δ (○) or WT (●) as a function of Mg²⁺ concentration. (A) Exchange rates (V_H⁺) were determined from experimental kinetics illustrated in Fig. 4 and described in Materials and Methods. Values are presented as mean V_H⁺ ± SE (n = 5). (B) Scatchard plot: K_m values derived from linear regression of data in A are 0.96 ± 0.02 mM and 0.92 ± 0.01 mM for vps5Δ and WT strains, respectively; V_max are 4.53 ± 0.36 and 2.93 ± 0.26%⋅min⁻¹ per mg of protein.

Figure 7

Ni²⁺ growth phenotypes of yeast mutants of the vacuolar protein-sorting pathway. Mutants were grown on agar/YNB minimal medium containing 0.1 mM Mg²⁺, with (+) or without (−) 0.2 mM Ni²⁺. Phenotypes were recorded after 4 days. Class (34) and name of each mutant are indicated. ? indicates class not determined or not available in the literature. Among these mutants, only Ni²⁺-resistant mutants were found to be defective in the retention of Golgi membrane proteins. We refer the reader to the Yeast Proteome Database (http://www.proteome.com) for further information about the mutants and the corresponding genes (including alternative names and functional details).