cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
The aim of this study was to investigate the effects of an overactivation of the cAMP/protein kinase A signaling pathway on the energetic metabolism of growing yeast. By using a cAMP-permeant mutant strain, we show that the rise in intracellular cAMP activates both anabolic and catabolic pathways. Indeed, different physiological patterns were observed with respect to the growth condition: (i) When cells were grown with a limiting amount of lactate, cAMP addition markedly increased the growth rate, whereas it only slightly increased the mitochondrial and cellular protein content. In parallel, the respiratory rate increased and the growth yield, as assessed by direct microcalorimetry, was not significantly modified by cAMP. (ii) Under conditions where the growth rate was already optimal (high lactate concentration), exogenous cAMP led to a proliferation of well-coupled mitochondria within cells and to an accumulation of cellular and mitochondrial proteins. This phenomenon was associated with a rise in the respiratory activity, thus leading to a drop in the growth yield. (iii) Under conditions of catabolic repression (high glucose concentration), cAMP addition markedly increased the fermentation rate and decreased the growth yield. It is concluded that overactivation of the cAMP/PKA pathway leads to uncoupling between biomass synthesis and catabolism, under conditions where an optimal growth rate is sustained by either a fermentative or a respiratory metabolism. D : S 0 0 0 5 -2 7 2 8 ( 0 2 ) 0 0 2 4 0 -2
2001
In the yeast Saccharomyces cerevisiae, three genes TPKl, TPK2, and TPK3 encode catalytic subunits of CAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPKl gene. In order to purify CI completely free of Cp and CS, a strain was constructed that contained only the TPKl gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPKl) and yeast regulatory subunit (BCYl) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-290, then bound to an antiregulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of CAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an M, of 52,000. Kinetic analysis showed that the apparent K,,, for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 PM, respectively. T...
Genes & Development, 1998
The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G 1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.
Comparative and Functional Genomics, 2004
We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30℃ to 50℃ on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway inSaccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the genePDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in thepde2Δ mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential facto...
TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae
Journal of general microbiology
Incubation of Succharomyces cereuisiae with the plant cytokinin W-(A2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpkl, tpk2, TPK3; tpkl, TPK2, tpk3) each deficient in two of the catalytic subunits of the CAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than CAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels. 0001-7508 O 1992 SGM
Journal of Biological Chemistry, 1988
In the yeast Saccharomyces cerevisiae, three genes TPKl, TPK2, and TPK3 encode catalytic subunits of CAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPKl gene. In order to purify CI completely free of Cp and CS, a strain was constructed that contained only the TPKl gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (T P K l) and yeast regulatory subunit (BCYl) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-290, then bound to an antiregulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of CAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an M, of 52,000. Kinetic analysis showed that the apparent K,,, for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 PM, respectively. The kcat was determined to be 640 min-l. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked. CAMP-dependent protein kinase has been shown to play an important role in the regulation of cellular processes (reviewed in Refs. 1-3). It is ubiquitous in eukaryotes from Dictyosteliurn discoideum and Saccharomyces cerevisiae to mammals. This enzyme serves as the link between hormonal stimulation of adenylate cyclase and a biological response. CAMP-dependent protein kinase functions to phosphorylate protein substrates thereby changing their biological properties. The best studied systems have been in mammalian liver, skeletal muscle, and adipocytes, in which CAMP-dependent protein kinase is involved in the regulation of the enzymes of glycogen and fatty acid metabolism. Recently, expression of several genes has been shown to be controlled by cAMP (4). These findings suggest that regulators of gene transcription may also be substrates of CAMP-dependent protein kinase (5). The inactive holoenzyme of CAMP-dependent protein ki-* This work was supported in part by National Institutes of Health Grant GM33986 (to M. J. Z.
Functional Interactions and Evolution of cAMP-PKA Signaling in Saccharomyces
2013
In an attempt to gain more insight on functional evolution of cAMP-PKA pathway I have taken a comparative approach and examined functional interactions of cAMP-PKA signaling in well-studied yeast developmental programs and closely related Saccharomyces sensu stricto species. I have shown that variation in cAMP-PKA signaling contributes significantly to variation in developmental responses in Saccharomyces cerevisiae. Variation in pseudohyphal growth and sporulation, two inversely correlated developmental strategies to nutrient limitation in yeast, proportional to variation in intracellular cAMP levels. S. cerevisiae strains proficient in pseudohyphal growth have higher intracellular cAMP concentrations relative to strains that sporulate efficiently. Phenotypic, genetic and signaling data presented here suggest that the cAMP-PKA signaling underlies a phenotypic trade-off between sporulation and pseudohyphal growth in S. cerevisiae. Further investigation into the role of cAMP-PKA signaling in closely related Saccharomyces paradoxus and Saccharomyces bayanus revealed an antagonistic function of cAMP-PKA signaling for developmental responses in S. bayanus. Unlike in S. cerevisiae, increased cAMP concentrations surprisingly inhibit pseudohyphal response in S. bayanus. Another unanticipated finding in this work is that in S. bayanus, Flo11, required for pseudohyphal differentiation in S. cerevisiae, is dispensable. Additionally, interactions of cAMP-PKA signaling and the general-stress response mechanism appear v reversed in S. bayanus. As shown by deletion mutation, gene expression and pharmacological treatment data, altered interactions and alternative targets downstream of cAMP-PKA could critically contribute to alternative regulation of nutrient-induced development in S. bayanus. Intracellular cAMP concentrations show decaying oscillations upon glucose replenishment in derepressed yeast cells. The quantitative characteristics of oscillations are distinct within and between Saccharomyces species. Given the tight regulation of cAMP levels and its critical role, the variation in cAMP oscillatory dynamics could be reflective of differential interactions of cAMP-PKA signaling that also underlie induction of developmental programs to changing environments. As such, intracellular cAMP levels and dynamics could potentially be used as molecular phenotypes.. vi Dedication I dedicate this thesis to my cherished grandparents, Cemile and Zeki Ural.
Yeast, 2017
Yeast cells can adapt their growth in response to the nutritional environment. Glucose is the favorite carbon source of Saccharomyces cerevisiae that prefers a fermentative metabolism despite the presence of oxygen. When glucose is consumed, the cell switches to the aerobic metabolism of ethanol, during the so-called diauxic shift. The difference between fermentative and aerobic growth is in part mediated by a regulatory mechanism called glucose repression. During glucose derepression a profound gene transcriptional reprogramming occurs and genes involved in the utilization of alternative carbon sources are expressed. Protein kinase A (PKA) controls different physiological responses following the increment of cAMP as a consequence of a particular stimulus. cAMP-PKA is one of the major pathways involved in the transduction of glucose signaling. In this work the regulation of the promoters of the PKA subunits during respiratory and fermentative metabolism are studied. It is demonstrated that all these promoters are upregulated in the presence of glycerol as carbon source through the Snf1/Cat8 pathway. However, in the presence of glucose as carbon source, the regulation of each PKA promoter subunits is different and only TPK1 is repressed by the complex Hxk2/Mig1 in the presence of active Snf1.