Project description:The conserved Snf1/AMPK (AMP-activated protein Kinase) family is one of the central components in nutrient sensing and regulation of carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic alpha-subunit Snf1, a regulatory gamma-subunit Snf4 and one of three possible beta-subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three beta-subunits by analyzing all 7 possible combinations of beta-subunit deletions together with the reference strain.
Project description:The conserved Snf1/AMPK (AMP-activated protein Kinase) family is one of the central components in nutrient sensing and regulation of carbon metabolism in eukaryotes. It is also involved in several other processes such as stress resistance, invasive growth and ageing. Snf1 kinase is composed of a catalytic α-subunit Snf1, a regulatory γ-subunit Snf4 and one of three possible β-subunits, Sip1, Sip2 or Gal83. We used a systematic approach to study the role of the three β-subunits by analyzing all 7 possible combinations of β-subunit deletions together with the reference strain.
Project description:The extent to which carbon flux is directed towards fermentation vs. respiration differs between cell types and environmental conditions. Understanding the basic cellular processes governing carbon flux is challenged by the complexity of the metabolic and regulatory networks. To reveal the genetic basis for natural diversity in channeling carbon flux, we applied Quantitative Trait Loci analysis by phenotyping and genotyping hundreds of individual F2 segregants of budding yeast that differ in their capacity to ferment the pentose sugar xylulose. Causal alleles were mapped to the RXT3 and PHO23 genes, two components of the large Rpd3 histone deacetylation complex. We show that these allelic variants modulate the expression of SNF1/AMPK-dependent respiratory genes. Our results suggest that over close evolutionary distances, diversification of carbon flow is driven by changes in global regulators, rather than adaptation of specific metabolic nodes. Such regulators may improve the ability to direct metabolic fluxes for biotechnological applications. mRNA profiles of S. cerevisiae strain BY4741 with either the RXT3 or PHO23 genes either deleted, replaced by S. cerevisiae T73 allele or replaced by S. cerevisiae PHO23 allele
Project description:The extent to which carbon flux is directed towards fermentation vs. respiration differs between cell types and environmental conditions. Understanding the basic cellular processes governing carbon flux is challenged by the complexity of the metabolic and regulatory networks. To reveal the genetic basis for natural diversity in channeling carbon flux, we applied Quantitative Trait Loci analysis by phenotyping and genotyping hundreds of individual F2 segregants of budding yeast that differ in their capacity to ferment the pentose sugar xylulose. Causal alleles were mapped to the RXT3 and PHO23 genes, two components of the large Rpd3 histone deacetylation complex. We show that these allelic variants modulate the expression of SNF1/AMPK-dependent respiratory genes. Our results suggest that over close evolutionary distances, diversification of carbon flow is driven by changes in global regulators, rather than adaptation of specific metabolic nodes. Such regulators may improve the ability to direct metabolic fluxes for biotechnological applications.
Project description:The yeast Snf1p/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1p regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1p is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the inositol polyphosphate kinase Kcs1p, which principally generates pyrophosphorylated forms of InsP7 and InsP8. Here, we report that Snf1p regulates Kcs1p, affecting Kcs1p phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1p phosphorylation, and Kcs1p is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1p directly phosphorylates Kcs1p, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP7, comparable to InsP7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing glycerol and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1p phosphorylation of Kcs1p, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth.
Project description:Sak1 (orf19.3840) of Candida albicans was found to be a kinase which phosphorylates and thereby activates Snf1, a highly conserved regulator of nutrient stress responses. Accordingly, a sak1 deletion mutant failed to grow on many alternative (i.e., non-glucose) carbon sources, but also showed a filamentation defect upon growth under glucose limitation and exhibited reduced virulence. To better define the effects of Sak1 on C. albicans metabolic adaptations, these transcriptional analyses were performed in complex medium, using a sak1 deletion mutant in comparison to the wild type.
Project description:In Saccharomyces cerevisiae, glycogen and trehalose are important reserve carbohydrates that accumulate under nutrient limitation in batch cultures. An inherent draw-back of batch studies is that specific growth rate and substrate and product concentrations are variable over time and between cultures. The aim of this present study was to identify the nutritional requirements associated with high accumulation of reserve carbohydrates at a fixed specific growth rate (0.10 h-1) in anaerobic chemostat cultures that were limited by one of five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc). Reserve carbohydrates accumulation is not a general response to nutrient limitation. Over the conditions tested, accumulation occurs essentially under nitrogen (and to a lesser extent carbon) limited conditions. This was confirmed by the transcriptional profile of the genes involved in trehalose biosynthesis. We show that the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4. However, the main regulatory control of glycogen biosynthesis was post-translational. Under nitrogen limitation, the ratio of glycogen synthase over glycogen phosphorylase increased up to eight-fold, thus enabling an increased flux towards glycogen biosynthesis.
Project description:In Saccharomyces cerevisiae, glycogen and trehalose are important reserve carbohydrates that accumulate under nutrient limitation in batch cultures. An inherent draw-back of batch studies is that specific growth rate and substrate and product concentrations are variable over time and between cultures. The aim of this present study was to identify the nutritional requirements associated with high accumulation of reserve carbohydrates at a fixed specific growth rate (0.10 h-1) in anaerobic chemostat cultures that were limited by one of five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc). Reserve carbohydrates accumulation is not a general response to nutrient limitation. Over the conditions tested, accumulation occurs essentially under nitrogen (and to a lesser extent carbon) limited conditions. This was confirmed by the transcriptional profile of the genes involved in trehalose biosynthesis. We show that the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4. However, the main regulatory control of glycogen biosynthesis was post-translational. Under nitrogen limitation, the ratio of glycogen synthase over glycogen phosphorylase increased up to eight-fold, thus enabling an increased flux towards glycogen biosynthesis. Experiment Overall Design: We studied this in anaerobic chemostat cultures at a dilution rate of 0.10 h-1 where growth was limited by five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc limitations). In addition, we studied the expression of these pathways at transcriptional and post-transcriptional levels and assessed the role of Msn2/4 in mediating transcriptional induction of glycogen and trehalose genes in the absence of stress.
Project description:Despite being the frontline therapy for Type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill-defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB. But several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for the translational response to metformin.
Project description:Despite being the frontline therapy for Type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill-defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB. But several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new RaptorAA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for the transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment.