Mitochondrial respiratory mutants of Saccharomyces cerevisiae accumulate glycogen and readily mobilize it in a glucose-depleted medium (original) (raw)
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FEMS Yeast Research, 2013
The Saccharomyces cerevisiae strain CBS6412 has been shown to be able to grow in synthetic medium containing glycerol as the sole carbon source, conditions under which laboratory strains such as CEN.PK and S288c cannot grow. Nonetheless, this strain exhibits a lag phase of c. 30-40 h following transition to glycerol medium. As mitochondria play a critical role in the dissimilation of the respiratory carbon source glycerol, we investigated mitochondrial function and dynamics throughout the lag phase using mitochondria-targeted roGFP, a redox-sensitive GFP variant. We found that following transition to glycerol medium, mitochondria become more oxidizing, accumulate near the bud neck, and exhibit decreased inheritance into daughter cells. Directly preceding entry into exponential growth phase, mitochondria become more reducing, mitochondrial accumulations at the bud neck decrease, and inheritance of mitochondria into daughter cells is restored.
Eukaryotic Cell, 2003
D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucosephosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu 3 Man 9 GlcNac 2 effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5⌬ or alg6⌬, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER).
Journal of Proteomics, 2014
The yeast Saccharomyces cerevisiae is a facultative aerobe able to adapt its metabolismaccording to the carbon substrate. The mechanisms of these adaptations involve at leastpartly the mitochondria but are not yet well understood. To address the possible role ofprotein phosphorylation event in their regulation, it is necessary in a first instance todetermine precisely the phosphorylation sites that show changes depending on the carbonsource. In this aim we performed an overall quantitative proteomic and phosphoproteomicstudy of isolated mitochondria extracted from yeast grown on fermentative (glucose orgalactose) and respiratory (lactate) media.Label free quantitative analysis of proteinaccumulation revealed significant variation of 176 mitochondrial proteins including 108proteins less accumulated in glucose medium than in lactate and galactose media. We alsoshowed that the responses to galactose and glucose are not similar. Stable isotope dimethyllabeling allowed the quantitative comparison of phosphorylation levels between the differentgrowth conditions. This study enlarges significantly the map of yeast mitochondrialphosphosites as 670 phosphorylation sites were identified, ofwhich 214 were new andquantified. Above all, we showed that 90 phosphosites displayed a significant variation according to the medium and that variation of phosphorylation levelis site-dependent. Biological significance This proteomic and phosphoproteomic study is the first extensive study providing quantitativedata on phosphosites responses to different carbon substrates independent of the variations ofprotein quantities in the yeastS. cerevisiaemitochondria. The significant changes observed in the level of phosphorylation according to the carbon substrate open the way to the study of theregulation of mitochondrial proteins by phosphorylation in fermentative and respiratory media. In addition, the identification of a large number of new phosphorylation sites show thatthe characterization of mitochondrial phosphoproteome is not yet completed.
European Journal of Biochemistry, 1988
The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3 -5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26"C, but were much more pronounced at the nonpermissive temperature of 35°C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyrl), able to incorporate exogenous cyclic AMP.
Glycogen phosphorylase activity in permeabilized cells of Saccharomyces cerevisae
Biochimica et Biophysica Acta (BBA) - Enzymology, 1974
The velocity of formation of glucose 6-phosphate and glucose by protamine-treated permeabilized yeast cells was dependent on the concentration of inorganic phosphate. Fluoride inhibited the reaction. Only small amounts of glucose 6-phosphate were produced in presence of ATP and glucose. We conclude that we are measuring the glycogen phosphorylase reaction inside the permeabilized cells.
Microbiology-sgm, 1985
Cells of Saccharomjces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro-fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondria1 cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium.
Applied and Environmental Microbiology, 2011
The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.
Applied and Environmental Microbiology, 2011
The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.