Assay of vacuolar pH in yeast and identification of acidification-defective mutants.

Abstract

As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification.Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo.Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vphl-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pepl2::LEU2 mutant appeared defective by fluorescence-ratio microscopy and qulnacrineuptake assays, but the vacuolar pH in thepepl2::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.Vacuoles in Saccharomyces cerevisiae are analogous to lysosomes of other organisms. Both types of organelles contain similar sets of proteases and other hydrolytic enzymes (1, 2), and both are energized and acidified by similar membrane-bound, proton-translocating ATPases (H+-ATPase) (3-6). Vacuolar proteases are required for intracellular protein turnover induced by nitrogen starvation (7). Apart from that typically "lysosomal" activity, a variety of additional functions have been ascribed to the vacuole, including maintenance of cytosolic amino acid homeostasis (8, 9), regulation of cellular calcium metabolism (8, 10), and others (11). Genetic analysis of protease-deficient mutants has been instrumental in delimiting the physiological significance of the vacuolar proteases (7 METHODSStrains and Culture Conditions. The wild-type strain was the diploid formed by mating strains X2180-1A and X2180-1B. Mutant strains and genotypes were BJ4895, a/a vphl-J/ vphl-1 trpl/+ leu2/+ +/ura3-52 and BJ4984, a/a pepl2:: LEU2/pepl2::LEU2 ura3-52/ura3-52 leu2-1/leu2-1 hisl1+ ade6/+. Growth media YPD and SC have been described (12). Cells in the logarithmic-growth phase were prepared by overnight growth in YPD or SC (10 ml) at 30'C in roller tube cultures. Culture densities were maintained below 2 x 107 cell per ml by dilution with YPD or SC as required.Labeling with Fluorescent Dyes. Medium for labeling with 6-CF diacetate (6-CFDA; C1362; Molecular Probes) was prepared by adding 50 mM citric acid to YPD, adjusting the pH to 3.0 with 12 M HCl, autoclaving, and then filtering (0.2 gm membrane filter); 6-CFDA was added to the medium immediately before use by dilution from 5 mM stock solutions in dimethyl sulfoxide; unless otherwise noted, the final 6-CFDA concentration was 5 1LM. Logarithmic-phase cells were harvested by membrane filtration, resuspended at 2 X 107 cell per ml in the labeling medium, and incubated with shakin...

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe

“…3A). Microscopic observation of vacuolar acidification with quinacrine fluorescence [26] showed a complete inhibition of vacuolar acidification of S. pombe cells by 10 lM concanamycin A (data not shown), suggesting no active transport of 2-DG into vacuoles.…”

A Genomic Screen for Yeast Vacuolar Membrane ATPase Mutants

“…V-ATPase subunit genes have fication by V-ATPases is important for receptor-ligand been identified through a variety of biochemical apuncoupling in the biosynthetic and endocytic pathways, proaches (Nelson et al 1989(Nelson et al , 1994 Beltran et zymogen activation, and cellular pH homeostasis. Supekova et al 1995) and genetic screens pH gradient generated by the V-ATPases can energize (Preston et al 1989;Ohya et al 1991;Ho et al 1993). uptake of Ca 2ϩ , amino acids, and other ions and metaboAlthough loss of V-ATPase activity in higher eukarylites (Nelson and Harvey 1999;Nishi and Forgac otes is lethal, disruption of any of the VMA genes in 2002).…”

“…The yeast V-ATPase has emerged mentable carbon sources, or in the presence of a variety as the predominant model system for eukaryotic V-ATPof heavy metals. Vph1p was identified through a screen ases because it closely resembles the enzyme in higher for vacuolar acidification defects (Preston et al 1989; eukaryotes but is more easily manipulated genetically Manolson et al 1992). Deletion of VPH1, which enand biochemically (Nelson and Klionsky 1996; Kane codes the vacuole-specific a-subunit isoform, results in and Smardon 2003).…”

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…Inactivation of any of the genes encoding V-ATPase subunits so far identified in S. cere isiae, with the exception of Vph1p, leads to loss of V-ATPase function. The resulting phenotype is not lethal, but there is a failure to grow in medium buffered to pH 7.5, sensitivity to high extracellular calcium concentrations and a dysfunctional vacuole [54][55][56][57][58][59][60][61] Other genes giving rise to the same phenotype [62] are present that encode polypeptides not yet identified as components of the V-ATPase, but which could possibly be involved in assembly, targeting or regulation of the enzyme. In addition, genes have been found that encode polypeptides with sequence similarity to identified V-ATPase subunits but whose function is not yet clear (see section 5.2.…”

“…An equivalent, if somewhat smaller, polypeptide is encoded by the I-gene in the Ntp gene cluster of Enterococcus [53]. A corresponding yeast 96 kDa protein was cloned by complementation of the ph1 mutation [54] and found to have 42 % identity with the rat protein [147], and all 116 kDa-related proteins sequenced to date appear to share a bipartite structure, with extensive N-terminal hydrophilic domain and a C-terminal domain containing up to seven putative membrane-spanning hydrophobic segments. The membrane topology of the protein remains unclear, but its sensitivity to proteolysis has led to the suggestion that the Nterminal domain is cytoplasmically exposed.…”