To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305-325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382-388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V 1 with V 0 , although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k cat for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance. Vacuolar Hϩ -ATPases (V-ATPases) 1 are found throughout the endomembrane system of all eukaryotes and at the plasma membrane of certain cells (1-4). In all of these locations, VATPases act as electrogenic proton pumps, coupling hydrolysis of cytosolic ATP to proton transport either into membranebound compartments or across the plasma membrane to the outside of the cell. V-ATPases consist of a peripheral complex containing the sites of ATP hydrolysis, the V 1 sector, attached to an integral membrane complex that forms the proton channel, the V 0 sector.Saccharomyces cerevisiae has proven to be an excellent model system for V-ATPases (1, 5-7). The yeast V-ATPase is very similar to those of higher eukaryotes both in its overall structure and in the primary sequence of its subunit genes. To date, 13 subunits have been identified in the yeast V-ATPase and shown to have homologues in other organisms. The V 1 sector contains eight subunits, designated A-H, which are encoded by the yeast VMA1, VMA2, VMA5, VMA8, VMA4, VMA7, VMA10, and VMA13 genes, respectively. The V 0 sector contains five subunits, designated a, c, cЈ, cЉ, and d, which are encoded by the VPH1 (or STV1), VMA3, VMA11, VMA16, and VMA6 genes, respectively (1).The mechanism by which the V-ATPase couples hydrolysis of cytosolic ATP to proton translocation is not fully understood. The F 1 F 0 -ATP synthase (F-ATPase) has been proposed as a model for the catalytic mechanism of V-ATPases (4). V-and F-ATPases are evolutionarily related, based on sequence similarity in the catalytic and regulatory nucleotide binding and the proteolipid subunits (8). Structural data for the enzymes also suggest they are related. Electron microscopic analysis of the V-ATPase demonstrates that, similar to the structure of the F-ATPase, the V 1 sector is attached to the V 0 sector through two stalks (9, 10...

The vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H ؉ -ATPase activity in Saccharomyces cerevisiae. The VMA41 gene was cloned and shown to be allelic to the CYS4 gene. The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells. cys4 mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidification in vivo. The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable. Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. Biochemical studies on the enzyme isolated from bovine clathrin-coated vesicles have indicated that reversible sulfhydryl-disulfide bond interconversion within the catalytic subunit may play a role in controlling V-ATPase activity in vivo (16 -18). Specifically, these studies show that disulfide bond formation between conserved cysteine residues near the nucleotide-binding site of the catalytic subunit results in inactivation of the V-ATPase and that this inactivation can be reversed by a disulfide interchange within the catalytic subunit. Furthermore, Dschida and Bowman (23) showed that reducing agents have a stabilizing effect on the V-ATPase from Neuro-* This work was supported in part by National Institutes of Health Grant R01-GM50322 and National Science Foundation Presidential Young Investigator Award MCB-9296244 (to P. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ American Heart Association Established Investigator. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Health Science Center at Syracuse, 750 E. Adams St., Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8750; Email: kanepm@vax.cs.hscsyr.edu.1 The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; V 1 , peripheral sector of the yeast vacuolar H ϩ -ATPase; V 0 , integral membrane sector of the yeast vacuolar H

Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma ؊ (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources. By screening based on these phenotypes and the inability of vma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. The VMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2 gene, which encodes a serine endoprotease localized to the late Golgi compartment. Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma ؊ growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo. However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from a kex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells. The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity. This is the first time a mutation of this type has been identified.A distinct class of proton-translocating ATPases, the vacuolar-type ATPases (V-ATPases), is responsible for acidifying the eukaryotic vacuolar network, including the vacuole or lysosome, Golgi apparatus, endosomes, clathrin-coated vesicles, and regulated secretory vesicles (9). The Saccharomyces cerevisiae vacuole is an acidic organelle functionally equivalent to the mammalian lysosome and the plant vacuole (50). It is involved in metabolite storage, macromolecular degradation, and calcium and amino acid homeostasis (2). The yeast V-ATPase is a multisubunit enzyme consisting of at least 12 different polypeptides encoded by the VMA genes (2, 27, 63; reference 15 and references therein). As in F 1 F 0 -ATPases, the enzyme is made up of two domains in the yeast V-ATPase: the peripheral sector (the V 1 sector), which contains the catalytic ATP-hydrolyzing domain and is peripherally associated with the cytoplasmic face of the vacuolar membrane, and the integral membrane sector (the V 0 sector), which contains the proton pore (27).Many of the genes that encode the yeast V-ATPase subunits, including VPH1, STV1 (a functional homolog of VPH1), VMA1 to -8, -10, -11, and -13, have been cloned (4,14,22,23,38,39,44,58,63,66,69). In addition, four genes which are required for assembly of the V-ATPase but are not part of the final active complex (VMA12, VMA21, VMA22, and VPH6) have be...

<b><i>Introduction:</i></b> Sodium zirconium cyclosilicate (SZC) is a selective potassium (K⁺) binder for hyperkalemia management that provides rapid and sustained correction of hyperkalemia. The NEUTRALIZE study is investigating whether SZC, in addition to correcting hyperkalemia and maintaining normal serum K⁺, can provide sustained increases in serum bicarbonate (HCO₃⁻) in patients with hyperkalemia and metabolic acidosis associated with chronic kidney disease (CKD). <b><i>Methods:</i></b> This is a prospective, randomized, double-blind, placebo-controlled phase 3b study of US adults with stage 3–5 CKD not on dialysis with hyperkalemia (K⁺ &#x3e;5.0–≤5.9 mmol/L) and low-serum HCO₃⁻ (16–20 mmol/L). In the open-label correction phase, all eligible patients receive SZC 10 g three times daily for up to 48 h. Patients who achieve normokalemia (K⁺ ≥3.5–≤5.0 mmol/L) are then randomized 1:1 to once-daily SZC 10 g or placebo for a 4-week, double-blind, placebo-controlled maintenance phase. The primary endpoint is the proportion of patients with normokalemia at the end of treatment (EOT) without rescue therapy for hyperkalemia. Key secondary endpoints include mean change in serum HCO₃⁻, the proportion of patients with an increase in serum HCO₃⁻ of ≥2 or ≥3 mmol/L without rescue therapy for metabolic acidosis, and the proportion of patients with serum HCO₃⁻ ≥22 mmol/L at EOT. <b><i>Conclusions:</i></b> NEUTRALIZE will establish whether SZC can provide sustained increases in serum HCO₃⁻ while lowering serum K⁺ in patients with hyperkalemia and CKD-associated metabolic acidosis and may provide insights on the mechanism(s) underlying the increased serum HCO₃⁻ with SZC treatment.