Presence Of Nonfermentable Carbon Source Research Articles

Exploring carbon source related localization and phosphorylation in the Snf1/Mig1 network using population and single cell-based approaches.

The AMPK/SNF1 pathway governs energy balance in eukaryotic cells, notably influencing glucose de-repression. In S. cerevisiae, Snf1 is phosphorylated and hence activated upon glucose depletion. This activation is required but is not sufficient for mediating glucose de-repression, indicating further glucose-dependent regulation mechanisms. Employing fluorescence recovery after photobleaching (FRAP) in conjunction with non-linear mixed effects modelling, we explore the spatial dynamics of Snf1 as well as the relationship between Snf1 phosphorylation and its target Mig1 controlled by hexose sugars. Our results suggest that inactivation of Snf1 modulates Mig1 localization and that the kinetic of Snf1 localization to the nucleus is modulated by the presence of non-fermentable carbon sources. Our data offer insight into the true complexity of regulation of this central signaling pathway in orchestrating cellular responses to fluctuating environmental cues. These insights not only expand our understanding of glucose homeostasis but also pave the way for further studies evaluating the importance of Snf1 localization in relation to its phosphorylation state and regulation of downstream targets.

Deletion of Voltage-Dependent Anion Channel 1 knocks mitochondria down triggering metabolic rewiring in yeast.

The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ0 cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a "branched" fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

Ime1 and Ime2 Are Required for Pseudohyphal Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

Pseudohyphal growth and meiosis are two differentiation responses to nitrogen starvation of diploid Saccharomyces cerevisiae. Nitrogen starvation in the presence of fermentable carbon sources is thought to induce pseudohyphal growth, whereas nitrogen and sugar starvation induces meiosis. In contrast to the genetic background routinely used to study pseudohyphal growth (Σ1278b), nonfermentable carbon sources stimulate pseudohyphal growth in the efficiently sporulating strain SK1. Pseudohyphal SK1 cells can exit pseudohyphal growth to complete meiosis. Two stimulators of meiosis, Ime1 and Ime2, are required for pseudohyphal growth of SK1 cells in the presence of nonfermentable carbon sources. Epistasis analysis suggests that Ime1 and Ime2 act in the same order in pseudohyphal growth as in meiosis. The different behaviors of strains SK1 and Σ1278b are in part attributable to differences in cyclic AMP (cAMP) signaling. In contrast to Σ1278b cells, hyperactivation of cAMP signaling using constitutively active Ras2(G19V) inhibited pseudohyphal growth in SK1 cells. Our data identify the SK1 genetic background as an alternative genetic background for the study of pseudohyphal growth and suggest an overlap between signaling pathways controlling pseudohyphal growth and meiosis. Based on these findings, we propose to include exit from pseudohyphal growth and entry into meiosis in the life cycle of S. cerevisiae.

Carnitine acetyltransferases are required for growth on non-fermentable carbon sources but not for pathogenesis in Candida albicans

Carbon starvation is a significant stress encountered by the opportunistic fungal pathogen Candida albicans, and mutations in several pathways required to assimilate non-fermentable carbon sources attenuate virulence. These pathways -- beta-oxidation, the glyoxylate cycle and gluconeogenesis -- are compartmentalized in the fungal cell between the peroxisome, mitochondria and cytosol; thus, the cell must transport key intermediates between these organelles. Transport of acetyl-CoA, a particularly important intermediate of carbon metabolism, is catalysed by membrane-associated carnitine acetyltransferases (CATs). We report here the characterization of the three predicted CAT genes in C. albicans, CTN1, CTN2 and CTN3. Strains lacking CTN1 or CTN2 were unable to grow on ethanol or acetate as sole carbon source; additionally, citrate was utilized poorly (Deltactn2) or not at all (Deltactn1) and the Deltactn2 mutant failed to grow on fatty acids as well. In contrast, deletion of CTN3 had no observable phenotype. All three genes were upregulated in the presence of non-fermentable carbon sources and after macrophage phagocytosis. CTN1 and CTN3 were able to complement the corresponding Saccharomyces cerevisiae Deltayat1 and Deltayat2 mutants. However, these mutants had no obvious attenuation in virulence in a mouse model of disseminated candidiasis, in contrast to other carbon metabolism mutants. These findings extend our understanding of nutrient stress in vivo and in vitro and the contribution of metabolic pathways to virulence in C. albicans.

Expression of the HXT13, HXT15 and HXT17 genes in Saccharomyces cerevisiae and stabilization of the HXT1 gene transcript by sugar-induced osmotic stress

Saccharomyces cerevisiae contains a family of 17 hexose transporter (HXT) genes; only nine have assigned functions, some of which are still poorly defined. Despite extensive efforts to characterize the hexose transporters, the expression of HXT6 and HXT8-17 remains an enigma. In nature, S. cerevisiae finds itself under extreme nutritional conditions including sugars in excess of 40% (w/v), depletion of nutrients and extremes of both temperature and pH. Using HXT promoter-lacZ fusions, we have identified novel conditions under which the HXT17 gene is expressed; HXT17 promoter activity is up-regulated in media containing raffinose and galactose at pH 7.7 versus pH 4.7. We demonstrated that HXT5, HXT13 and, to a lesser extent, HXT15 were all induced in the presence of non-fermentable carbon sources. HXT1 encodes a low-affinity transporter and in short-term osmotic shock experiments, HXT1 promoter activity was reduced when cells were exposed to media containing 40% glucose. However, we found that the HXT1 mRNA transcript was stabilized under conditions of osmotic stress. Furthermore, the stabilization of HXT1 mRNA does not appear to be gene specific because 30 min after transcriptional arrest there is a fourfold more mRNA in osmotically stressed versus non-stressed yeast cells. A large portion of S. cerevisiae mRNA molecules may, therefore, have a decreased rate of turnover during exposure to osmotic stress indicating that post-transcriptional regulation plays an important role in the adaptation of S. cerevisiae to osmotic stress.

Identification in Saccharomyces cerevisiae of Two Isoforms of a Novel Mitochondrial Transporter for 2-Oxoadipate and 2-Oxoglutarate

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.

Extragenic suppressors of yeast glucose derepression mutants leading to constitutive synthesis of several glucose-repressible enzymes

Saccharomyces cerevisiae regulatory genes CAT1 and CAT3 constitute a positive control circuit necessary for derepression of gluconeogenic and disaccharide-utilizing enzymes. Mutations within these genes are epistatic to hxk2 and hex2, which cause defects in glucose repression. cat1 and cat3 mutants are unable to grow in the presence of nonfermentable carbon sources or maltose. Stable gene disruptions were constructed inside these genes, and the resulting growth deficiencies were used for selecting epistatic mutations. The revertants obtained were tested for glucose repression, and those showing altered regulatory properties were further investigated. Most revertants belonged to a single complementation group called cat4. This recessive mutation caused a defect in glucose repression of invertase, maltase, and iso-1-cytochrome c. Additionally, hexokinase activity was increased. Gluconeogenic enzymes are still normally repressible in cat4 mutants. The occurrence of recombination of cat1::HIS3 and cat3::LEU2 with some cat4 alleles allowed significant growth in the presence of ethanol, which could be attributed to a partial derepression of gluconeogenic enzymes. The cat4 complementation group was tested for allelism with hxk2, hex2, cat80, cid1, cyc8, and tup1 mutations, which were previously described as affecting glucose repression. Allelism tests and tetrad analysis clearly proved that the cat4 complementation group is a new class of mutant alleles affecting carbon source-dependent gene expression.

Identification and characterization of a thermolabile antigen (TLAa, enolase) in Saccharomyces cerevisiae.

Five kinds of proteins of Saccharomyces cerevisiae were recently purified to a homogeneous state and identified as yeast cell surface antigens (TLAa, TLAb, TLAc, TLAd, and TLAe), but their physiological functions remained uncertain. In this paper, TLAa was identified as a yeast enolase (EC 4.2.1.11) from the following evidence: (1) molecular weights and amino acid compositions of both proteins were similar, (2) the N-terminal 22 amino acid sequences of both proteins were the same (3), TLAa had enolase activity, and (4) the authentic yeast enolase gave a positive reaction with anti-TLAa serum in the Ouchterlony immunodiffusion test and the immunoblotting test. Two isoproteins of TLAa (enolase) were separated by non-denatured polyacrylamide gel electrophoresis and detected by immunoblotting with anti-TLAa serum. The contents of the two isoproteins varied depending on the following culture conditions: glucose starvation, growth in the presence of non-fermentable carbon sources, and growth in media containing sodium chloride and 2-deoxyglucose. The contents did not vary, however, with heat shock treatment or with growth in media containing sodium azide, tunicamycin, or sorbitol. These results showed that TLAa was a cytoplasmic antigen, and that its synthesis was regulated by some environmental stresses.

Demarcation of a sequence involved in mediating catabolite repression of the gene for the 11 kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae.

A regulatory element has been identified in the promoter region of the gene encoding the 11 kDa subunit VIII of the ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae. The element, which is approximately 40 bp long and situated 185 bp upstream of the initiator ATG, is essential for induction of gene expression during growth in the presence of non-fermentable carbon sources. This is shown by the regulated synthesis of beta-galactosidase in yeast cells harbouring a CYC1-lacZ fusion gene, in which the CYC1 UAS's had been replaced by a 43 bp subunit VIII gene promoter fragment. In addition two DNA-binding activities, which may represent either separate factors or different forms of a single factor, have been detected. Both factors are abundant and they bind in a mutually exclusive fashion to a DNA sequence just upstream of the regulatory element. Although it is unlikely that these factors are directly involved in the response of the subunit VIII gene to catabolite repression, the position of their binding sites relative to the UAS and to the 3'-terminus of a gene located only 361 bp upstream suggest that they are important in modulating transcriptional activity of this region.

Effects of temperature on nucleo-mitochondrial interactions in the yeast [formula omitted

The temperature, as well as several antibacterial antibiotics could be used to differentiate mitochondrial protein synthesis (MPS) from the cytoplasmic (CPS) one in the yeast Saccharomyces cerevisiae . In fact MPS and CPS have respectively the optimum at 30°C and 36°C. A series of cellular processes, as the mitotic reproduction in presence of non-fermentable carbon sources, the synthesis of galactose pathway enzymes and the meiotic process have the same optimal temperature (30°C), whereas the growth of the wild type in presence of fermentable carbon sources and of a galactose repressor constitutive mutant (i-) have the optimal temperature of 36°C, in agreement with our previous hypothesis in which the expression of some sections of the nuclear genetic complement is dependent on regulatory functions controlled by MPS.