Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+ -ATPase.

The Ca2+ -ATPase homolog of Saccharomyces cerevisiae, PMR1, cloned by Rudolph et al. (Cell 58 (1989) 133-145) is required for normal Golgi functions. We have investigated the role of Pmr1-protein in maintaining homeostasis of cytosolic free Ca2+ concentration ([Ca2+]i). It was found that exposure to moderately high Ca2+ concentrations led to elevated levels of [Ca2+]i in cells of pmr1 null mutant, in comparison with cells of pmr2 isogenic mutant (defective in cell-membrane Na+ - ATPase) and of an isogenic wild type. In addition, we showed that PMR1 deletion causes massive accumulation of Ca2+ in the vacuoles and affects the rates of Ca2+ influx and efflux.

Calcium homeostasis in yeast cells exposed to high concentrations of calcium. Roles of vacuolar H(+)-ATPase and cellular ATP.

Cytosolic Ca2+ concentrations ([Ca2+]i) were determined in haploid and diploid cells of Saccharomyces cerevisiae, loaded with indo-1 and exposed to media containing a range of Ca2+ concentrations. [Ca2+]i homeostasis was maintained at the 100-150 nM level in cells which were pre-incubated with glucose and exposed to 0.1 microM-10 mM Ca2+ in the medium. Slightly higher levels of [Ca2+]i were determined in cells exposed to 50 mM Ca2+. Pre-incubation with metabolic inhibitors instead of glucose caused a reduction in cellular ATP levels and an impaired [Ca2+]i homeostasis; [Ca2+]i reached 800 nM in cells exposed to 10 mM CaCl2. Cells of the delta vma4 mutant strain, with no functional vacuolar H(+)-ATPase, had elevated levels of [Ca2+]i, reaching 1.8 microM when pre-incubated with glucose and exposed to 10 mM CaCl2. Higher levels of [Ca2+]i were measured in the mutant cells which were pre-incubated with metabolic inhibitors. This result indicates the central role of the vacuoles in maintaining [Ca2+]i-homeostasis and suggests the presence of an additional non-vacuolar ATP-requiring mechanism which contributes to keeping [Ca2+]i at low levels.

An intracellular ATP-dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3.

We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP-dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP-dependent Ca2+ accumulation into non-vacuolar Ca(2+)-storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P-type ATPases. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400-18407] strongly reduced the level of ATP-dependent Ca2+ uptake into non-vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP-dependent Ca2+ pump. The residual ATP-dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca(2+)-ATPase encoded by a different gene.

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels.

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium homeostasis and transport are affected by disruption of cta3, a novel gene encoding Ca2(+)-ATPase in Schizosaccharomyces pombe.

A new P-type ATPase gene, cta3, has been identified in Schizosaccharomyces pombe. The deduced amino acid sequence presents a 45% identity with the Saccharomyces cerevisiae putative Ca2(+)-ATPase encoded by the PMR2 gene. The cta3 protein contains 7 out of the 8 amino acid residues involved in high affinity Ca2+ binding in the sarcoplasmic reticulum Ca2(+)-ATPase from muscles. It also contains a region similar to the phospholamban-binding domain that characterizes this Ca2+ pump. A null mutation of cta3 leads to higher levels of cytosolic free Ca2+ and to lower amounts of sequestered and bound Ca2+. Cellular Ca2+ efflux and rates of uptake into intracellular compartments are reduced by the loss of cta3 function. The sequence analysis and the physiological results strongly support the conclusion that the cta3 gene encodes a Ca2(+)-ATPase, probably located in intracellular membranes.

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1.

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.

Distinction of species and strains of mycoplasmas (mollicutes) by genomic DNA fingerprints with an rRNA gene probe.

Genomic fingerprints of Acholeplasma laidlawii, Mycoplasma hominis, and Mycoplasma pneumoniae strains were obtained by Southern blot hybridization of the digested mycoplasmal DNAs with an rRNA gene probe. The hybridization patterns revealed genotypic heterogeneity among A. laidlawii and M. hominis strains and a remarkable degree of homogeneity among M. pneumoniae strains isolated from pneumonia patients during a 10-year period. Genomic fingerprints with the rRNA gene probe can thus serve as indicators of intraspecies genetic homogeneity or heterogeneity and can provide a new, sensitive tool for strain identification with a potential for application in epidemiology.

Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae.

Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry.