Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: Intracellular pH and the Effect of Membrane Depolarizing Compounds

Thevelein, Johan M.; Beullens, Monique; Honshoven, F; Hoebeeck, G; Detremerie, K; Hollander, Jan A. den; Jans, A. W. H.

doi:10.1099/00221287-133-8-2191

Cited by 65 publications

(78 citation statements)

References 13 publications

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“…Early 31 P NMR studies indicated that yeast intracellular pH was not affected by the external pH conditions or the presence of NH 4 ϩ (41). However, several subsequent studies found that the intracellular pH of yeast followed the external pH when the extracellular medium was either strongly buffered (18,(42)(43)(44) or contained weak organic acids such as acetate, butyrate, or succinate (19,(45)(46)(47). This suggests that the plasma membrane of yeast is permeable not only to NH 4 ϩ but also to other weak acids.…”

Section: Nh 4 ϩ -Mediated Acidification Of the Yeast Vacuolementioning

confidence: 99%

Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast

Plant¹,

Manolson²,

Grinstein³

et al. 1999

Journal of Biological Chemistry

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Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (VATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking VATPase activity (⌬vma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When ⌬vma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H ؉ -ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the ⌬vma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wildtype and the ⌬vma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH 4 ؉ , routinely added as a nitrogen source, was present. The NH 4 ؉ -dependent acidification was not due to efflux of NH 3 from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH 4 ؉ . These findings suggest that although NH 3 can act as a cell-permeant proton scavenger, NH 4 ؉ may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH 4 ؉ frequently added as a nitrogen source to yeast culture media together with effective NH 4 ؉ transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.Acidification of defined endomembrane compartments along the endocytic and secretory pathways is essential for cellular function. Intraorganellar acidification appears to control vesicular traffic (1) as well as receptor ligand dissociation within endosomes (1). Similarly, acidification is thought to be essential for protein degradation in lysosomes (2) and for microbial elimination in phagosomes (3). In yeast and plants acidification of the vacuole provides the driving force for secondary transport of a variety of ions and metabolites (4 -7).Acid equivalents are concentrated in the lumen of these organelles by active H ϩ pumping mediated by the vacuolartype ATPase (V-ATPase), 1 an evolutionarily conserved multimeric enzyme (see Refs. 8 -11 for review). Considering the array of critical cellular functions that depend on the acidification generated by the V-ATPase, this enzyme would be predicted to be e...

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Section: Nh 4 ϩ -Mediated Acidification Of the Yeast Vacuolementioning

confidence: 99%

Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast

Plant¹,

Manolson²,

Grinstein³

et al. 1999

Journal of Biological Chemistry

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“…For cAMP determination, cells were grown on YP medium supplemented with 3 '/o glycerol and 0.1 % galactose until lateexponential phase. Incubation with glucose and fructose (100 mM each) and quantification of cAMP followed our established protocol (Thevelein et al, 1987). The same culturing regime was used for cells in which the levels of glycolytic metabolites were determined according to Novel yeast hexokinase mutations de Koning & van Dam (1992).…”

Section: Methodsmentioning

confidence: 99%

Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2

et al. 1999

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In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxkl), hexokinase PI1 (Hxk2) and a specific glucokinase (Glkl). The authors have shown previously that either Hxkl or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression). Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxkl or Hxk2 is sufficient for long-term repression by fructose. Mutants lacking the TPSl gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In t h i s study, suppressor mutations of the growth defect of a t p s l A h x k l A double mutant on fructose were isolated and identified as novel HXK2 alleles. All six alleles studied have single amino acid substitutions. The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms. The mutations conferred different effects on long-and shortterm repression. Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity. The data show that the long-and short-term phases of catabolite repression can be dissected using different hexokinase mutations. The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression. Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short-or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression.

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“…Up to now, only two triggers of the yeast Ras-adenylate cyclase pathway have been identified : glucose and related fermentable sugars (Van der Plaat, 1974;Mbonyi et al, 1988) and intracellular acidification (Thevelein et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-medium-induced (FGM) pathway

et al. 1997

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In cells of the yeast Sacchammyces cerevisiae, trehalase activation, repression of CTTl (catalase), SSA3 (Hsp7O) and other STRE-controlled genes, feedback inhibition of CAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by CAMP but requires catalytic activity of CAMP-dependent protein kinase (cAPK; Tpkl, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in wiwo. In witm measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two-to threefold, in both the absence and presence of CAMP. In wiwo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by deletion of SCHS is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCHS abolished the responses of the protein kinase A targets in glucoserepressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of m l and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.

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Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: Intracellular pH and the Effect of Membrane Depolarizing Compounds

Cited by 65 publications

References 13 publications

Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast

Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast

Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2

The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-medium-induced (FGM) pathway

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