Academic literature on the topic 'Saccharomyces cerevisiae, cell cycle, Snf1'

ABSTRACT Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G1.

ABSTRACT In Saccharomyces cerevisiae, RDS2 encodes a zinc cluster transcription factor with unknown function. Here, we unravel a key function of Rds2 in gluconeogenesis using chromatin immunoprecipitation-chip technology. While we observed that Rds2 binds to only a few promoters in glucose-containing medium, it binds many additional genes when the medium is shifted to ethanol, a nonfermentable carbon source. Interestingly, many of these genes are involved in gluconeogenesis, the tricarboxylic acid cycle, and the glyoxylate cycle. Importantly, we show that Rds2 has a dual function: it directly activates the expression of gluconeogenic structural genes while it represses the expression of negative regulators of this pathway. We also show that the purified DNA binding domain of Rds2 binds in vitro to carbon source response elements found in the promoters of target genes. Finally, we show that upon a shift to ethanol, Rds2 activation is correlated with its hyperphosphorylation by the Snf1 kinase. In summary, we have characterized Rds2 as a novel major regulator of gluconeogenesis.

Abstract Pho85, a protein kinase with significant homology to the cyclin-dependent kinase, Cdc28, has been shown to function in repression of transcription of acid phosphatase (APase, encoded by PHO5) in high phosphate (Pi) medium, as well as in regulation of the cell cycle at G1/S. We describe several unique phenotypes associated with the deletion of the PHO85 gene including growth defects on a variety of carbon sources and hyperaccumulation of glycogen in rich medium high in Pi. Hyperaccumulation of glycogen in the pho85 strains is independent of other APase regulatory molecules and is not signaled through Snf1 kinase. However, constitutive activation of cAPK suppresses the hyperaccumulation of glycogen in a pho85 mutant. Mutation of the type-1 protein phosphatase encoded by GLC7 only partially suppresses the glycogen phenotype of the pho85 mutant. Additionally, strains containing a deletion of the PHO85 gene show an increase in expression of GSY2. This work provides evidence that Pho85 has functions in addition to transcriptional regulation of APase and cell-cycle progression including the regulation of glycogen levels in the cell and may provide a link between the nutritional state of the cell and these growth related responses.

SUMMARY The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states. Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients. This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health. Here, we review our knowledge of one “model” quiescent cell population, in cultures of yeast grown to stationary phase in rich media. We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state. We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle. We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways. We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis. Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available.

ABSTRACTHypoxia has critical effects on the physiology of organisms. In the yeastSaccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by anSNF1knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia,S. cerevisiaecells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol viade novoprotein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

ABSTRACT In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. ConstitutiveADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressingADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells.

ABSTRACT Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of ∼40% of these genes, including a prominent subclass involved in nutrient acquisition, is not understood. To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter, PHO5. We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2. PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate. Polyphosphate levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation. Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation. Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3Δ strain. Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion. These results demonstrate a dynamic mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through the cell cycle.

Abstract The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.

The AMP-activated protein kinase (AMPK) family is a group of Serine/Threonine kinases highly conserved in eukaryotes, from yeast and insects to plants and mammals. Their primary role is the integration of signals regarding nutrient availability and environmental stresses, ensuring the adaptation to those conditions and cell survival (Hardie G., 2007; Ghillebert R. et al., 2011). As its homologue AMPK, in Saccharomyces cerevisiae Snf1 exists as a heterotrimeric complex. Core of this enzyme is the catalytic α subunit (Snf1), made up of a canonical catalytic domain in its N-terminus and of an autoinhibitory C-terminal domain which mediates the interaction with the regulatory subunits of this kinase (Rudolph M.J. et al., 2005). These subunits are: the β subunit (Sip1, Sip2 and Gal83, alternatively), which regulates Snf1 localization (Vincent O. et al., 2000) and the γ subunit (Snf4) that, interacting with the autoinhibitory domain of Snf1, guarantees the complete activation of the kinase (Momcilovic M. et al., 2008). Beyond the interaction with Snf4, the activation of the protein kinase Snf1 is determined by the phosphorylation of Thr210 residue in the α subunit (McCartney R.R. and Schmidt M.C., 2001). Three upstream kinases (Sak1, Tos3, Elm1) are responsible for such a phosphorylation. Those kinases are constitutively active, but metabolic signals, such as high glucose concentrations, promote the activity of the phosphatase complex Reg1/Glc7 which dephosphorylates and hence inactivates Snf1 (Huang D. et al.,1996; Sanz P. et al., 2000; Sutherland C. et al., 2003; Hong S. et al., 2003). In budding yeast, Snf1 is required for adaptation to glucose limitation and for growth on non-fermentable carbon sources. In those conditions Snf1 controls the expression of more than 400 genes. Apart from carbon metabolism, Snf1 affects several other processes; in fact, this kinase controls the expression of some important genes involved in the resistance to different environmental stresses (osmotic and alkaline stresses) or in the regulation of different cellular processes such as sporulation, aging, filamentous and invasive growth (Portillo F. et al., 2005; Ashrafi K. et al., 2000; Vyas V.K. et al., 2003). As a transcriptional regulator, Snf1 exerts its role modulating gene transcription at different levels. This kinase regulates different transcription factors, such as the transcription inhibitor Mig1 (Treitel M.A. et al., 1998; Papamichos-Choronaris M. et al., 2004) or some other transcription factors like Adr1, Sit4, Cat8 and Gcn4 which regulate the expression of genes involved in central metabolic functions, such as gluconeogenesis and respiration (Hedbacker K. and Carlson M., 2008; Smets B. et al., 2010; Kacherovsky N. et al., 2008). Moreover, protein kinase Snf1 is even able to positively regulate the transcription of some metabolic genes influencing the chromatin remodelling process and the recruitment of some Pre-Initiation Complex (PIC) components at promoters. In fact, Snf1 promotes acetylation of histone H3 by either the direct phosphorylation of Ser10 of histone H3 and the phosphorylation of acetyl-tranferase Gcn5 (Lo W. et al., 2005; van Oevelen C.J. et al., 2006; Liu Y. et al., 2010). Moreover, Snf1 is involved in the recruitment to some promoters of Mediator complex (Young E.T. et al., 2002), SAGA complex (van Oevelen C.J. et al., 2006), TATA-binding protein (TBP) (Shirra M.K. et al., 2005) and RNA Pol II (Tachibana C. et al., 2007; Young R.T. et al., 2012). My PhD research activity was focused on the role of Snf1 in the regulation of the expression of G1-specific genes, and thus in its function as modulator of cell cycle progression. Data obtained in our laboratory showed that, in cells grown in 2% glucose, deletion of SNF1 gene caused a delayed G1/S phase transition, consistently with a decreased expression of CLB5 gene. In keeping with that defective expression, the snf1Δ strain showed a severe reduction of Clb5 protein levels and a consequent decrease of phosphorylation of Clb5/Cdk1 complex targets, such as Sld2, which are responsible for the onset of DNA replication. Moreover, our co-immunoprecipitation assays highlighted that Snf1 interacts with Swi6, the common subunit of SBF (Swi4-Swi6) and MBF (Mbp1-Swi6) transcription complexes which regulate the expression of G1-specific genes (Nasmyth, K. and Dirick, L., 1991; Koch C. et al., 1993). Remarkably, the phenotype of the snf1 null mutant was complemented by a glucose concentration higher than 2% (5%), suggesting that the role of Snf1 in the modulation of cell cycle progression could depend on the nutritional status of cells. Those data, published in Pessina S. et al., 2010, newly indicated that Snf1 was involved in the regulation of G1/S transition and pointed to a role for this kinase in the modulation of G1-specific gene expression. To gain further insight into the function of Snf1, we then analyzed the expression profile of G1-specific genes in cells synchronized in G1 phase by α-factor treatment and released into fresh medium. Our analyses showed that loss of Snf1 (snf1Δ strain) severely affected the expression of CLN2, PCL1 (SBF-dependent) and CLB5, RNR1 (SBF-dependent) genes, suggesting that Snf1 regulates the expression of both SBF- and MBF-dependent genes. Although protein Snf1 was not detectable at promoters of G1-specific genes, we investigated whether it could modulate the activity of SBF and MBF complexes and we found that in a snf1Δ strain the recruitment of Swi6 to G1-specific promoters was affected. Moreover, our Chromatin ImmunoPrecipitation (ChIP) assays also showed that in a snf1Δ strain the defective association of Swi6 to promoters led to a decreased recruitment of both the FACT complex, which is involved in the chromatin remodelling at G1-specific promoters, and of the RNA Pol II. Since it is known that the subcellular localization of Swi6 influences its interaction with promoters, then we analyzed its localization in G1 synchronized cells. In keeping with literature data (Sidorova J.M. et al., 1995; Taberner, F.J. and Igual, J.C., 2010), our analyses showed that in wild type cells synchronized in G1 phase by α-factor treatment Swi6 was essentially nuclear. Instead, in a snf1Δ strain Swi6 was localized in the nucleus only in the 60% of the G1-arrested cells, consistently with the reduced binding of Swi6 to G1-specific promoters. It is well known that the Swi6 interaction to DNA is mediated by the DNA binding-proteins Swi4 and Mbp1 (Andrews B.J. and Moore L.A., 1992; Moll T. et al., 1992). Therefore we extended our analyses to those proteins and we found that also the nuclear localization and the subsequent binding to DNA of Swi4 and Mbp1 were affected in a snf1Δ strain. Therefore, our data provide a representative snapshot of what occurs in vivo in a snf1 null mutant, supporting the notion that Snf1 promotes the expression of G1-specific genes modulating the nuclear localization of SBF and MBF components and thus promoting the formation of a complete Pre-initiation Complex (PIC) at G1-specific promoters. It is well known that phosphorylation of Snf1 at Thr210 leads to the full activation of the kinase (Hong S.P. et al., 2003; Sutherland C.M. et al., 2003). Then, in order to obtain insight into the Snf1 molecular mechanism in cell cycle regulation, we investigated its phosphorylation on Thr210 during cell cycle progression. Snf1 was slightly phosphorylated on the Thr210 residue during all the cell cycle, suggesting that this kinase was partially active. To determine whether the activation of Snf1 was involved in its function as regulator of G1 transcription, we analyzed the expression of SBF- and MBF-dependent genes in the SNF1-T210A mutant and we found that in this mutant the expression of those genes was reduced. In keeping with those data, the expression of G1-specific genes resulted affected also in a SNF1-K84R mutant, in which the ATP binding site has been destroyed causing a severe reduction of Snf1 kinase activity. On the base of those findings, we investigated whether Snf1 could exert its role in G1 phase through the phosphorylation of specific substrates and we found that Snf1 phosphorylates in vitro Swi6 on Ser760. Nevertheless, analyses of site-specific mutants (SWI6-S760A or SWI6-S760E) did not show any alteration of G1/S transition, suggesting that this phosphorylation was not involved in the role of Snf1 as regulator of cell cycle. The ChIP analyses of Swi6 binding to CLN2 and RNR1 promoters, then, showed that in the SNF1-K84R mutant the recruitment of Swi6 was slightly affected; nevertheless, that alteration was not severe as that of a snf1Δ strain. Consistently, neither the recruitment of FACT complex nor the binding of RNA Pol II to G1-specific promoters was affected in the SNF1-K84R mutant. Since this last finding seemed to disagree with the severe reduction of mRNA expression of SBF- and MBF-dependent genes observed in the SNF1-K84R mutant, we wondered whether defects in transcriptional elongation might occur in that strain. Thus, we analyzed the occupancy of FACT complex and of RNA Pol II at the internal regions of CLN2 and RNR1 genes by ChIP analyses; in the SNF1-K84R mutant the occupancy of both those complexes was decreased, suggesting that the kinase activity of Snf1 promotes the transcriptional elongation across G1-specific genes. In conclusion, the sum of data here presented indicates that protein kinase Snf1 is involved at different levels in the modulation of the G1-specific gene expression, thus highlighting a new function for Snf1 in the regulation of G1/S transition.

Snf1 è una serina/treonina chinasi necessaria per il lievito S. cerevisiae per la crescita in condizioni di limitazione di nutrienti e per l’utilizzo di fonti di carbonio alternative al glucosio. Nel nostro laboratorio è stato precedentemente dimostrato che la mancanza di Snf1 causa un difetto nella transizione G1/S del ciclo cellulare e un difetto nell’espressione dei geni di fase G1 anche in condizioni di sufficienza nutrizionale (2% glucosio). È stato quindi approfondito il coinvolgimento di Snf1 in tre importanti processi cellulari: ciclo, trasduzione del segnale e metabolismo. Per dimostrare la necessità dell’attività catalitica di Snf1 per una corretta transizione G1/S è stato utilizzato un ceppo Snf1-I132G, in cui l’attività della chinasi può essere inibita utilizzando l’inibitore specifico 2NM-PP1. Il difetto nell’effettuare la transizione G1/S e nella trascrizione dei geni di fase G1 di questo ceppo in presenza dell’inibitore è stata dimostrata sia con esperimenti di rilascio da α-factor sia mediante elutriazione. Nello studio del coinvolgimento di Snf1 nella regolazione di altri pathway di trasduzione del segnale è stata identificata, mediante esperimenti di CoIP/MS, l’interazione tra Snf1 e l’adenilato ciclasi (Cyr1), l’enzima responsabile della produzione di AMP ciclico (cAMP), attivatore di PKA. Il dominio della proteina Cyr1 contenente il RAS Associating Domain e 2 putativi siti consenso di Snf1 è stato purificato in E.coli e ne è stata dimostrata la fosforilazione in vitro da parte della chinasi. È stato inoltre da dimostrato che in un ceppo Snf1-G53R, in cui la chinasi è costitutivamente attivata, si ha una riduzione di circa il 50% nel contenuto di cAMP intracellulare, assieme alla deregolazione dell’espressione di geni PKA-dipendenti. É stata quindi ipotizzata l’esistenza di un crosstalk fra i pathway di Snf1 e PKA. Per chiarire il ruolo globale di Snf1 in condizioni di sufficienza nutrizionale è stata effettuata un’analisi trascrittomica (gene-chip) di cellule wt e snf1∆ cresciute in 2% e 5% glucosio. É stato così evidenziato che la mancanza di Snf1 in 2% glucosio, ma non in 5%, causa la deregolazione di circa 1000 geni, fra i quali ad esempio i geni glicolitici. Sono pertanto state indagate le deregolazioni metaboliche presenti in cellule prive di Snf1. L’analisi dei metaboliti secreti da cellule snf1Δ in 2% glucosio ha permesso di dimostrare che, in relazione alla propria velocità di crescita, producono più etanolo ed acetato in confronto a cellule wt. Questa attività glicolitica maggiore del wt è abolita, coerentemente a quanto già osservato, in presenza di 5% glucosio. É stato quindi dimostrato che anche nelle condizioni di crescita dei nostri esperimenti cellule snf1Δ presentano un accumulo di acidi grassi, fenotipo già osservato con bassi livelli di glucosio e dovuto all’assenza di fosforilazione Snf1-dipendente dell’enzima acetil-CoA carbossilasi. Una più estesa analisi metabolica, sia mediante spettrometria di massa che mediante NMR, ha permesso di descrivere in dettaglio i riarrangiamenti metabolici che cellule snf1Δ subiscono perché la crescita sia garantita nonostante i processi anabolici sopra descritti. Cellule snf1Δ in 2% glucosio accumulano glutammato in funzione di un maggiore consumo degli amminoacidi forniti nel terreno, evento necessario per il mantenimento della velocità di crescita del mutante. Il mutante inoltre accumula intermedi del ciclo degli acidi tricarbossilici e se trattato con antimicina A, un inibitore del della catena di trasporto degli elettroni, subisce un effetto deleterio in 2%, ma non in 5% glucosio. Il trattamento influenza negativamente crescita e contenuto di ATP e causa nel mutante aumento di NADH, dimostrandone la mancata riossidazione mitocondriale.
Snf1 is a serine/threonine kinase required by the yeast S. cerevisiae to grow in nutrient-limited conditions and to utilize carbon sources alternative to glucose. In our laboratory we previously demonstrated that lack of Snf1 causes an impairment of the G1/S transition of the cell cycle and a defect in the expression of genes of the G1 phase, even in condition of glucose sufficiency (2% glucose). It was therefore investigated the involvement of Snf1 in three important cellular processes: cycle, signal transduction and metabolism. To demonstrate the necessity of the catalytic activity of Snf1 for a proper G1/S transition was utilized a Snf1-I132G strain, in which the catalytic activity of the kinase can be inhibited by the specific inhibitor 2NM-PP1. The impairment of the G1/S transition and of the transcription of G1 genes in this strain in the presence of the inhibitor was demonstrated performing α-factor release and elutriation experiments. Studying the involvement of Snf1 in the regulation of other signaling pathways it was identified, through CoIP/MS experiments, the interaction between Snf1 and adenylate cyclase (Cyr1), the enzyme responsible for the synthesis of cyclic AMP (cAMP), activator of PKA. The RAS Associating Domain of Cyr1, containing 2 putative Snf1 phosphorylation sites, was purified in E. coli and its in vitro phosphorylation by Snf1 was demonstrated. Moreover, in a Snf1-G53R strain, in which the kinase is constitutively active, we found a reduction of 50% of intracellular cAMP, together with the deregulation of the expression of PKA-dependent genes. We therefore hypothesized the existence of a crosstalk between the Snf1 and PKA pathways.To investigate the global role of Snf1 in conditions of nutritional sufficiency we performed a transcriptomic analysis (gene chip) of wt and snf1Δ cells grown in 2% and 5% glucose, evidencing that lack of Snf1 causes the deregulation of about 1000 genes in 2%, but not in 5% glucose. Among these there are glycolytic genes and therefore possible metabolic deregulations in the absence of Snf1 were investigated. snf1Δ cells grown in 2% glucose secrete more ethanol and acetate, in proportion to their growth rate, compared to the wt. This enhanced glycolytic activity is abolished, as observed for transcripts, in 5% glucose. We further demonstrated that in our growth condition snf1Δ cells accumulate fatty acids, as previously observed in low glucose, due to the lack of Snf1-dependent phosphorylation of acetyl-CoA carboxylase. An extended metabolic analysis, both through mass spectrometry and NMR, revealed in detail the metabolic rewiring occurring in snf1Δ cells to guarantee the growth in spite of the enhanced anabolic processes. snf1Δ cells in 2% glucose accumulate glutamate, coming from the degradation of supplemented amino acids, in an essential process to maintain the growth rate of the mutant. Moreover, the mutant accumulates TCA cycle intermediates and in 2%, but not 5% glucose, is negatively affected by treatment with antimycin A, inhibitor of the electron transport chain. The treatment impairs growth and ATP content and increases NADH in the mutant, demonstrating the necessity of its mitochondrial reoxidation.

Multiple checkpoint controls ensure that later cellular events are not initiated until previous cellular events have been successfully completed. Our laboratory studies the checkpoint at the G2/M boundary that ensures the integrity of chromosome transmission by blocking mitosis until DNA synthesis and repair is completed. The checkpoint-dependent cell division arrest is one of several prominent responses to DNA damage, which also includes transcriptional induction of damage-inducible genes and DNA repair. I undertook three projects that explore several aspects of the damage response: (1) I further characterized the checkpoint gene RAD24, in that I showed that RAD24 function has G2 phase-specificity after damage and that RAD24 contributes to genomic stability; (2) I evaluated the nature of the damage signal from UV-irradiation that elicits a checkpoint-dependent cell cycle arrest; and (3) I established a transcriptional role for some of the checkpoint genes. In addition, I characterized a gene that encodes a novel elongation factor-type GTPase. Upon examination of the checkpoint-dependent delay following UV-irradiation in a mutant defective for incision of pyrimidine dimers, I found that processing of DNA damage, i.e. dimer-incision, is necessary to generate an appropriate damage signal. Processing of damage may be a general property of the damage response and may involve the checkpoint proteins. I found that some checkpoint genes have an additional role in a complex transcriptional induction response to DNA damage. Primarily, I found: (i) mec1 and mec2 mutants are defective for DNA damage-induction of the RNR3 gene, whereas the other checkpoint mutants appear to play less of a role; (ii) all the checkpoint mutants are proficient for transcriptional induction UBI4; (iii) rad17 mutants, and to a lesser degree mec1 and mec2 mutants as well, are defective for damage-induction of DDR48; (v) transcription of the RAD17, RAD24, MEC1, and MEC2 (but not the RAD9) checkpoint genes is damage-inducible and MEC1 is required for the transcriptional induction of the MEC1 and MEC2 genes, but not the RAD17 or RAD24 genes. I suggest that their transcriptional function ties the checkpoint proteins to DNA repair, as damage-inducible transcriptional induction probably functions to augment repair.

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.

Le cycle cellulaire eucaryote est caractérisé par des changements abrupts et dynamiques de la polarité cellulaire lorsque les chromosomes sont dupliqués et ségrégés. Ces évènements nécessitent une coordination entre la machinerie du cycle cellulaire et les régulateurs de la polarité. Les mécanismes qui contrôlent cette coordination ne sont pas totalement compris. Dans la levure S. cerevisiae, comme dans d’autres organismes eucaryotes, la GTPase Cdc42 joue un rôle important dans la régulation de la polarité cellulaire. En effet ses régulateurs constituent un module GTPase qui subit une phosphorylation dynamique, au cours du cycle cellulaire, par des kinases évolutivement conservées dont la Cycline-Dependent Kinase 1 (Cdk1) et la p21-Activated Kinase (PAK). Ces kinases et substrats pourraient relier la polarité et la progression dans le cycle cellulaire. En utilisant une approche in vitro, nous avons reconstitué la phospho-régulation du Guanine nucléotide Exchange Factor (GEF) de Cdc42, la protéine Cdc24. Nous avons identifié un possible mécanisme de régulation de la phosphorylation impliquant une protéine d’échafaudage qui augmente la phosphorylation de Cdc24 par la PAK et Cdk1. Cette phosphorylation accroit modérément l’affinité de Cdc24 pour cette même protéine d’échafaudage, Bem1. De plus, en testant les effets d’autres composants du module GTPase sur la phosphorylation de Cdc24, nous avons identifié un effet antagoniste pour une GTPase Activating Protein (GAP), Rga2. Cette protéine est présente dans le même complexe que Cdc24 et Bem1, les membres de ce complexe sont tous phosphorylés par Cdk1. Des mutants rga2 suggèrent que la phosphorylation que subie Rga2 inhibe son activité GAP. Nous proposons un modèle provisoire pour expliquer la présence de Rga2 dans ce complexe et l’inhibition qu’elle oppose à la phosphorylation de Cdc24. La présence de la protéine GAP dans le complexe pourrait être un mécanisme de contrôle de la phosphorylation de Cdc24 dans le but de déstabiliser son intéraction avec la protéine Bem1 en cas de mauvaise localisation du complexe. Par ailleurs, la PAK est activée par l’activité de Cdc42, nos résultats sont consistants avec un modèle dans lequel des signaux du cycle cellulaire engendreraient une auto-amplification de l’activation du module GTPase. Chez S. pombe, la croissance polarisée nécessite un gradient d’activation de Cdc42 dû à une ségrégation de GEF et de GAP. Dans ces travaux nous montrons que toutes les protéines GAPs de Cdc42 localisent aux sites de croissance au cours du cycle cellulaire. Ces localisations sont consistantes avec le besoin de cyclage de Cdc42 pour maintenir sa polarisation. Ces résultats suggèrent que la localisation des protéines GAP régulant Cdc42 chez S. cerevisiae semble différente de ce qui est connu chez S. pombe
The eukaryotic cell cycle is characterized by abrupt and dynamic changes in cellular polarity as chromosomes are duplicated and segregated. Those dramatic cellular events require coordination between the cell cycle machinery and polarity regulators. The mechanisms underlying this coordination are not well understood. In the yeast S. cerevisiae, as in other eukaryotes, the GTPase Cdc42 plays an important role in the regulation of cell polarity. Cdc42 regulators constitute a GTPase module that undergoes dynamic phosphorylation during the cell cycle by conserved kinases including Cyclin-Dependent Kinase 1 (Cdk1) and p21-activated kinase (PAK). These kinases and substrates may link cell polarity to the cell cycle progression. Using in vitro approaches, we have reconstituted the phospho-regulation of the Cdc42 Guanine Nucleotide Exchange Factor (GEF), Cdc24. We have identified a possible mechanism of Cdc24 regulation involving a scaffold-dependent increase in Cdc24 phosphorylation by Pak and Cdk1. This phosphorylation moderately increases the affinity of Cdc24 for another GTPase module component, the scaffold Bem1. Moreover, by testing the effect of other GTPase module components on the phosphorylation of Cdc24, and thus on its interaction with the scaffold, we identified an antagonistic function for the GTPase Activating Protein (GAP) Rga2. Our in vivo data of rga2 mutants suggest that Rga2 phosphorylation by Cdk1 inhibits its GAP activity. We propose a tentative model to explain the inhibition of Cla4 by Rga2 and its presence in a complex containing Cdc24 and Bem1. The presence of the GAP protein in the complex may be a mechanism that reduces Cdc24 phosphorylation in case of a mistargetting of the complex in order to downregulate the GEF/Scaffold dimer. Since the PAK component of the GTPase module is itself activated by Cdc42 activity, our results are consistent with a model in which inputs from the cell cycle lead to auto-amplification of the Cdc42 GTPase module. In S. pombe, polarised growth requires a gradient of activation of Cdc42 due to GEF and GAP segregation. Here we show that all Cdc42 GAPs localise to the polarised site during the cell cycle. Those localisations are consistent with a requirement of Cdc42 cycling to maintain a polarity cap. Our results may suggest that Cdc42 GAPs localisations in S. cerevisiae are different from current knowledge in S. pombe