Search

The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallett et al., 2015) reported that AMPK in yeast, that is Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells. ispartof: Elife vol:12 pages:e84319- ispartof: location:England status: Published online

All proliferating cells need to match metabolism, growth and cell cycle progression with nutrient availability to guarantee cell viability in spite of a changing environment. In yeast, a signaling pathway centered on the effector kinase Snf1 is required to adapt to nutrient limitation and to utilize alternative carbon sources, such as sucrose and ethanol. Snf1 shares evolutionary conserved functions with the AMP-activated Kinase (AMPK) in higher eukaryotes which, activated by energy depletion, stimulates catabolic processes and, at the same time, inhibits anabolism. Although the yeast Snf1 is best known for its role in responding to a number of stress factors, in addition to glucose limitation, new unconventional roles of Snf1 have recently emerged, even in glucose repressing and unstressed conditions. Here, we review and integrate available data on conventional and non-conventional functions of Snf1 to better understand the complexity of cellular physiology which controls energy homeostasis.

We present an improved extraction and derivatization protocol for GC-MS analysis of amino/non-amino acids in Saccharomyces cerevisiae. Yeast cells were extracted with chloroform: aqueous-methanol (1:1, v/v) and the resulting non-polar and polar extracts combined and dried for derivatization. Polar and non-polar metabolites were derivatized using tert-butyldimethylsilyl (t-BDMS) dissolved in acetonitrile. Using microwave treatment of the samples, the derivatization process could be completed within 2 h (from >20 h of the conventional method), providing fully derivatized metabolites that contain multiple derivatizable organic functional groups. This results in a single derivative from one metabolite, leading to increased accuracy and precision for identification and quantification of the method. Analysis of combined fractions allowed the method to expand the coverage of detected metabolites from polar metabolites i.e. amino acids, organic acids and non-polar metabolites i.e. fatty alcohols and long-chain fatty acids which are normally non detectable. The recoveries of the extraction method was found at 88 ± 4%, RSD, N = 3 using anthranilic acid as an internal standard. The method promises to be a very useful tool in various aspects of biotechnological applications i.e. development of cell factories, metabolomics profiling, metabolite identification, 13C-labeled flux analysis or semi-quantitative analysis of metabolites in yeast samples.

Here we propose the optimization of a rapid and reproducible protocol for intracellular metabolite extraction from yeast cells and their metabolic profiling by (1)H-NMR spectroscopy. The protocol reliability has been validated through comparison between the metabolome of cells in different phases of growth or with different genetic backgrounds.

In Saccharomyces cerevisiae , the entrance into S phase requires the activation of a specific burst of transcription, which depends on SBF (SCB binding factor, Swi4/Swi6) and MBF (MCB binding factor, Mbp1/Swi6) complexes. CK2 is a pleiotropic kinase involved in several cellular processes, including the regulation of the cell cycle. CK2 is composed of two catalytic subunits (α and α′) and two regulatory subunits (β and β′), both of which are required to form the active holoenzyme. Here we investigate the function of the CK2 holoenzyme in Start-specific transcription. The ckb1 Δ ckb2 Δ mutant strain, bearing deletions of both genes encoding CK2 regulatory subunits, shows a delay of S-phase entrance due to a severe reduction of the expression of SBF- and MBF-dependent genes. This transcriptional defect is caused by an impaired recruitment of Swi6 and Swi4 to G 1 gene promoters. Moreover, CK2 α and β′ subunits interact with RNA polymerase II, whose binding to G 1 promoters is positively regulated by the CK2 holoenzyme. Collectively, these findings suggest a novel role for the CK2 holoenzyme in the activation of G 1 transcription.

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.

In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription.

Background Yeast cells have developed a variety of mechanisms to regulate the activity of metabolic enzymes in order to adjust their metabolism in response to genetic and environmental perturbations. This can be achieved by a massive reprogramming of gene expression. However, the transcriptional response cannot explain the complexity of metabolic regulation, and mRNA stability regulation, non-covalent binding of allosteric effectors and post-translational modifications of enzymes (such as phosphorylation, acetylation and ubiquitination) are also involved, especially as short term responses, all converging in modulating enzyme activity. Scope of review The functional significance of post-translational modifications (PTMs) to the regulation of the central carbon metabolism is the subject of this review. Major conclusions A genome wide analysis of PTMs indicates that several metabolic enzymes are subjected to multiple PTMs, suggesting that yeast cells can use different modifications and/or combinations of them to specifically respond to environmental changes. Glycolysis and fermentation are the pathways where phosphorylation, acetylation and ubiquitination are most frequent, while enzymes of storage carbohydrate metabolism are especially phosphorylated. Interestingly, some enzymes, such as the 6-phosphofructo-2-kinase Pfk26, the phosphofructokinases Pfk1 and Pfk2 and the pyruvate kinase Cdc19, are hubs of PTMs, thus representing central key regulation nodes. For the functionally better characterized enzymes, the role of phosphorylations and lysine modifications is discussed. General significance This review focuses on the regulatory mechanisms of yeast carbon metabolism, highlighting the requirement of quantitative, systematical studies to better understand PTM contribution to metabolic regulation.