ABSTRACT
Pseudomonas putida S12 is a solvent-tolerant gamma-proteobacterium with an extensive track record for production of industrially relevant chemicals. Here we report the annotated complete genome sequence of this organism, including the megaplasmid pTTS12 which encodes many of the unique features of the S12 strain.
Subject(s)
Genome, Bacterial , Pseudomonas putida/genetics , Molecular Sequence Data , Plasmids , SolventsABSTRACT
Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose-galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose-galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.
Subject(s)
Energy Metabolism , Galactose/metabolism , Gene Expression Regulation, Fungal , Glucose/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Aerobiosis , Anaerobiosis , Culture Media , Fermentation , Proteomics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Addition of ammonium sulphate to nitrogen-depleted yeast cells resulted in a transient increase in Ins(1,4,5)P(3), with a maximum concentration reached after 7-8 min, as determined by radioligand assay and confirmed by chromatography. Surprisingly, the transient increase in Ins(1,4,5)P(3) did not trigger an increase in the concentration of intracellular calcium, as determined in vivo using the aequorin method. Similar Ins(1,4,5)P(3) signals were also observed in wild-type cells treated with the phospholipase C inhibitor 3-nitrocoumarin and in cells deleted for the only phospholipase C-encoding gene in yeast, PLC1. This showed clearly that Ins(1,4,5)P(3) was not generated by phospholipase C-dependent cleavage of PtdIns(4,5)P(2). Apart from a transient increase in Ins(1,4,5)P(3), we observed a transient increase in PtdIns(4,5)P(2) after the addition of a nitrogen source to nitrogen-starved glucose-repressed cells. Inhibition by wortmannin of the phosphatidylinositol 4-kinase, Stt4, which is involved in PtdIns(4,5)P(2) formation, did not affect the Ins(1,4,5)P(3) signal, but significantly delayed the PtdIns(4,5)P(2) signal. Moreover, wortmannin addition inhibited the nitrogen-induced activation of trehalase and the subsequent mobilization of trehalose, suggesting a role for PtdIns(4,5)P(2) in nitrogen activation of the fermentable-growth-medium-induced signalling pathway.
Subject(s)
Calcium Signaling/physiology , Inositol 1,4,5-Trisphosphate/metabolism , Nitrogen/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Yeasts/metabolism , Ammonium Sulfate/pharmacology , Calcium/metabolism , Glucose/metabolism , Inositol 1,4,5-Trisphosphate/isolation & purification , Type C Phospholipases/metabolism , Yeasts/drug effectsABSTRACT
In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.
Subject(s)
Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/physiology , Proteins/metabolism , Saccharomyces cerevisiae Proteins , Actins/metabolism , Enzyme Activation , G2 Phase , Hydroxyurea/pharmacology , Intracellular Signaling Peptides and Proteins , Mitosis/physiology , Mutagenesis , Nocodazole/pharmacology , Peptidylprolyl Isomerase , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 2 , Protein Tyrosine Phosphatases/metabolism , Proteins/genetics , Proteins/physiology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Spindle Apparatus/physiologyABSTRACT
Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.
Subject(s)
Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits , Gene Expression Regulation, Fungal , Glucose/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae/metabolism , Signal Transduction , Biological Transport , Fungal Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hexokinase/metabolism , Hydrogen-Ion Concentration , Phosphorylation , Receptors, Cell Surface/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.
Subject(s)
Cyclic AMP/metabolism , Fungal Proteins/metabolism , GTP-Binding Protein alpha Subunits , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hexokinase/metabolism , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Signal Transduction , Amino Acid Substitution , Biological Transport , Carbohydrate Metabolism , Extracellular Space , Fructose/metabolism , Fructose/pharmacology , Glucose/pharmacology , Glucose-6-Phosphate/metabolism , Hexoses/metabolism , Monosaccharide Transport Proteins/metabolism , Phosphorylation , Saccharomyces cerevisiae/drug effectsABSTRACT
The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.
Subject(s)
Cell Cycle , Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , CDC28 Protein Kinase, S cerevisiae/metabolism , Cell Cycle/drug effects , Cyclins/genetics , Flow Cytometry , Fungal Proteins/genetics , G1 Phase/drug effects , Gene Deletion , Gene Expression Regulation, Fungal/drug effects , Genes, Fungal/genetics , Glucose/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Mating Factor , Meiosis/drug effects , Membrane Proteins , Peptides/pharmacology , Peptidylprolyl Isomerase , Phenotype , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2 , Proteins/genetics , RNA, Fungal/analysis , RNA, Fungal/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Signal Transduction/drug effects , Sirolimus/pharmacology , Spores, Fungal/cytology , Spores, Fungal/drug effects , Spores, Fungal/enzymology , Spores, Fungal/metabolism , Temperature , Tripeptidyl-Peptidase 1ABSTRACT
We have characterized a novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae. The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells. The N-terminal half of the Rgs2 protein consists of a typical RGS domain. Deletion and overexpression of Rgs2, respectively, enhances and reduces glucose-induced accumulation of cAMP. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes. Deletion of RGS2 causes opposite phenotypes. We demonstrate that Rgs2 functions as a negative regulator of glucose-induced cAMP signalling through direct GTPase activation of the Gs-alpha protein Gpa2. Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1. Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins.
Subject(s)
Fungal Proteins/antagonists & inhibitors , Fungal Proteins/metabolism , GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins , Heterotrimeric GTP-Binding Proteins/antagonists & inhibitors , RGS Proteins/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Cyclic AMP/metabolism , Down-Regulation , Fungal Proteins/genetics , GTP-Binding Protein alpha Subunits, Gq-G11 , Gene Deletion , Gene Expression , Genes, Fungal , Genetic Complementation Test , Glucose/pharmacology , Guanosine Triphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Hydrolysis , Mice , Molecular Sequence Data , Phenotype , RGS Proteins/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed.
Subject(s)
Cyclic AMP/metabolism , Hexokinase/metabolism , Saccharomyces cerevisiae/enzymology , Signal Transduction , Amino Acid Sequence , Base Sequence , Carbohydrate Metabolism , Catalysis , Cloning, Molecular , DNA Primers , Hexokinase/chemistry , Hexokinase/genetics , Lysine/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, stress resistance and proliferation, in particular in connection with the available nutrient conditions. Extensive information has been obtained on the core section of the pathway, i.e. Cdc25, Ras, adenylate cyclase, PKA, and on components interacting directly with this core section, such as the Ira proteins, Cap/Srv2 and the two cAMP phosphodiesterases. Recent work has now started to reveal upstream regulatory components and downstream targets of the pathway. A G-protein-coupled receptor system (Gpr1-Gpa2) acts upstream of adenylate cyclase and is required for glucose activation of cAMP synthesis in concert with a glucose phosphorylation-dependent mechanism. Although a genuine signalling role for the Ras proteins remains unclear, they appear to mediate at least part of the potent stimulation of cAMP synthesis by intracellular acidification. Recently, several new targets of the PKA pathway have been discovered. These include the Msn2 and Msn4 transcription factors mediating part of the induction of STRE-controlled genes by a variety of stress conditions, the Rim15 protein kinase involved in stationary phase induction of a similar set of genes and the Pde1 low-affinity cAMP phosphodiesterase, which specifically controls agonist-induced cAMP signalling. A major issue that remains to be resolved is the precise connection between the cAMP-PKA pathway and other nutrient-regulated components involved in the control of growth and of phenotypic characteristics correlated with growth, such as the Sch9 and Yak1 protein kinases. Cln3 appears to play a crucial role in the connection between the availability of certain nutrients and Cdc28 kinase activity, but it remains to be clarified which nutrient-controlled pathways control Cln3 levels.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits , Heterotrimeric GTP-Binding Proteins , Phosphoric Diester Hydrolases , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/genetics , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Adenylyl Cyclases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 1 , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Glucose/metabolism , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Signal Transduction , Transcription, Genetic , ras Proteins/metabolismABSTRACT
In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.
Subject(s)
Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Blotting, Northern , Cyclic AMP-Dependent Protein Kinases/metabolism , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Molecular Sequence Data , Mutation , Receptors, Cell Surface/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Signal TransductionABSTRACT
Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.
Subject(s)
Cyclic AMP/metabolism , Fungal Proteins/metabolism , GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/metabolism , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins , Saccharomyces cerevisiae/metabolism , Signal Transduction , ras Proteins/metabolism , Adenylyl Cyclases/metabolism , Cell Cycle Proteins/metabolism , Cyclic AMP/biosynthesis , Down-Regulation , Hydrogen-Ion Concentration , Phosphoprotein Phosphatases/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins , rap GTP-Binding Proteins , ras-GRF1ABSTRACT
We have used RNA fingerprinting by the mRNA Differential Display technique to identify new genes in the yeast Saccharomyces cerevisiae, expression of which is controlled by specific nutrient conditions. mRNA was isolated from cells grown on glucose medium into exponential and stationary phase, and from cells starved for nitrogen on glucose-containing medium. To avoid interference with the large number of glucose-repressible genes, a glucose-repression-deficient strain was used. Twenty different sets of arbitrary primers chosen at random were used for PCR-amplification of reverse transcriptase generated cDNAs, which resulted in six highly reproducible gene expression patterns. The validity of the approach was confirmed by sequencing PCR products of genes with known expression patterns, SUP44/RPS4, CTT1, SSA3, HSP30 and HSP104, and genes with related functions, TEF1 and TEF3, encoding translation elongation factors. In all cases the specificity of the responses was confirmed by Northern blot analysis. The results show that the PCR-mapping method is highly useful for the identification of new genes expressed under specific conditions in the yeast S. cerevisiae.
Subject(s)
Gene Expression Regulation, Fungal , Genes, Fungal , Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , RNA, Messenger/analysisABSTRACT
Addition of rapidly fermentable sugars to cells of the yeast Saccharomyces cerevisiae grown on nonfermentable carbon sources causes a variety of short-term and long-term regulatory effects, leading to an adaptation to fermentative metabolism. One important feature of this metabolic switch is the occurrence of extensive transcriptional repression of a large group of genes. We have investigated transcriptional regulation of the SUC2 gene encoding repressible invertase, and of HXK1, HXK2 and GLK1 encoding the three known yeast hexose kinases during transition from derepressed to repressed growth conditions. Comparing yeast strains that express various combinations of the hexose kinase genes, we have determined the importance of each of these kinases for establishing the catabolite-repressed state. We show that catabolite repression involves two distinct mechanisms. An initial rapid response is mediated through any kinase, including Glk1, which is able to phosphorylate the available sugar. In contrast, long-term repression specifically requires Hxk2 on glucose and either Hxk1 or Hxk2 on fructose. Both HXK1 and GLK1 are repressed upon addition of glucose or fructose. However, fructose repression of Hxk1 is only transient, which is in line with its preference for fructose as substrate and its requirement for long-term fructose repression. In addition, expression of HXK1 and GLK1 is regulated through cAMP-dependent protein kinase. These results indicate that sugar sensing and establishment of catabolite repression are controlled by an interregulatory network, involving all three yeast sugar kinases and the Ras-cAMP pathway.
Subject(s)
Carbohydrate Metabolism , Glucokinase/metabolism , Glycoside Hydrolases/metabolism , Hexokinase/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , DNA Primers/genetics , DNA, Fungal/genetics , Fermentation , Fructose/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Glucokinase/genetics , Glucose/metabolism , Glycoside Hydrolases/genetics , Hexokinase/genetics , Nitrogen/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction , Substrate Specificity , beta-FructofuranosidaseABSTRACT
Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex, GGS1/TPS1, TPS2, TPS3 and TSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth only TSL1 shows an expression pattern like that of the STRE-controlled genes CTT1 and SSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.
Subject(s)
Glucosyltransferases/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Blotting, Northern , Cell Division/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation , Enzyme Activation , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Glucose/metabolism , Glucosyltransferases/biosynthesis , Glucosyltransferases/metabolism , HSP70 Heat-Shock Proteins/genetics , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Regulatory Sequences, Nucleic Acid , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
Transcription of the QCR8 gene, encoding subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR), is controlled by the carbon-source-dependent heme-activator protein complex HAP2/3/4 and the general transcriptional regulators autonomous replication-site-binding factor ABF1 and centromere-binding and promoter-binding factor CPF1. In this study, we investigate and dissect the relative contributions and mutual interactions of these regulators in transcriptional control. Transcription was analyzed both under steady-state conditions and during nutritional shifts, in hap delta mutants and after site-specific mutagenesis of the various binding sites in the chromosomal context of the QCR8 gene. We present evidence for both direct and indirect interactions between ABF1 and HAP2/3/4, and show that HAP2/3/4 is essential for a rapid transcriptional induction during transition from repressed to derepressed conditions. However, the activator is not the only determinant for carbon-source-dependent regulation, and we observe a functional difference between HAP2/3/4 and the HAP2/3 subcomplex. ABF1 is required for maintainance of basal repressed and derepressed transcription in the steady state of growth. The repressive action of the negative modulator CPF1 during escape from glucose repression is overcome through the cooperative action of ABF1 and HAP2/3/4. The implications of the intricate interactions of these DNA-binding regulators for control of expression of mitochondrial protein genes are discussed.
Subject(s)
CCAAT-Binding Factor , Electron Transport Complex III/genetics , Genes, Fungal , Mitochondria/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , DNA, Fungal/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Glucose/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Promoter Regions, Genetic , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/drug effectsABSTRACT
The KlCPF1 gene, coding for the centromere and promoter factor CPF1 from Kluyveromyces lactis, has been cloned by functional complementation of the methionine auxotrophic phenotype of a Saccharomyces cerevisiae mutant lacking ScCPF1. The amino-acid sequences of both CPF1 proteins show a relatively-low overall identity (31%), but a highly-homologous C-terminal domain (86%). This region constitutes the DNA-binding domain with basic-helix-loop-helix and leucine-zipper motifs, features common to the myc-related transcription factor family. The N-terminal two-thirds of the CPF1 proteins show no significant similarity, although the presence of acidic regions is a shared feature. In KlCPF1, the acidic region is a prominent stretch of approximately 40 consecutive aspartate and glutamate residues, suggesting that this part might be involved in transcriptional activation. In-vitro mobility-shift experiments were used to establish that both CPF1 proteins bind to the consensus binding site RTCACRTG (CDEI element). In contrast to S. cerevisiae, CPF1 gene-disruption is lethal in K. lactis. The homologous CPF1 genes were transformed to both S. cerevisiae and K. lactis cpf1-null strains. Indistinguishable phenotypes were observed, indicating that, not withstanding the long nonconserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.
Subject(s)
Centromere , Gene Expression Regulation, Fungal , Kluyveromyces/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Binding Sites , Consensus Sequence , DNA-Binding Proteins/metabolism , Genes, Fungal , Genetic Complementation Test , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
The abundant DNA-binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well-defined chromatin in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite-repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease-hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady-state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.
Subject(s)
DNA-Binding Proteins/metabolism , Electron Transport Complex III/genetics , Genes, Fungal/genetics , Nucleosomes/ultrastructure , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Chromatin/ultrastructure , Fungal Proteins/metabolism , Micrococcal Nuclease/metabolism , Models, Genetic , Substrate SpecificitySubject(s)
Gene Expression Regulation, Fungal , Mitochondria/metabolism , Saccharomyces cerevisiae/metabolism , Base Sequence , Cell Cycle , DNA, Fungal/genetics , DNA-Binding Proteins/metabolism , Electron Transport , Energy Metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal , Heme/physiology , Models, Genetic , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Signal Transduction , Transcription, GeneticABSTRACT
The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two factors play in transcriptional regulation of this gene. To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter. The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts. We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication. CPF1 acts as a negative regulator, modulating the overall induction response. Alleviation of repression through CPF1 requires passage through the S phase. Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.