Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Pugh, Tom; Shah, Jyoti C.; Magee, Patrick; Clancy, Mary J.

doi:10.1016/0167-4838(89)90294-x

Cited by 26 publications

(24 citation statements)

References 62 publications

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“…S. cerevisiae has three genes known to encode hydrolytic glucoamylases, SGA1, STA1, and STA2. The STA genes are exoenzymes thought to be involved in the utilization of starch as a nutrient (59), whereas SGA1 encodes a very similar enzyme reported to be sporulation-specific and associated with the vacuole (9,48). Lack of GPH1 eliminated the transient drop in glycogen in the early stationary phase and resulted in an exaggerated resynthesis phase (Fig.…”

Section: Resultsmentioning

confidence: 99%

Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

Wang

Wilson

Fujino

et al. 2001

Molecular and Cellular Biology

273

242

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In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.Cells constantly abstract information about their environment and modify their cellular and metabolic programs to cope with the prevailing conditions. For unicellular organisms like the budding yeast, Saccharomyces cerevisiae, much of the information about nutritional status is carried by the nutrients themselves. Depending on the type and availability of carbon, nitrogen, sulfur, and other requirements, the appropriate metabolic and cellular programs are elicited. Exhaustion of a preferred carbon source, like glucose, signals the induction of numerous genes needed to change to other metabolic regimes, in part by derepression of glucose-repressed genes and in part by cyclic AMP (cAMP) pathway control of gene expression (18,30). Another decision linked to nutritional deprivation is the synthesis of storage compounds like glycogen and trehalose, which are the primary carbohydrate reserves of the yeast (15). Glycogen is a branched polymer of glucose units that acts as a reserve of glucose and energy (47). In yeast growing on glucose, glycogen is synthesized late in the logarithmic phase and begins to be utilized when cells enter the stationary phase (7,8,37). However, glycogen stores can be p...

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Section: Resultsmentioning

confidence: 99%

Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

Wang

Wilson

Fujino

et al. 2001

Molecular and Cellular Biology

273

242

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“…The SGA was purified by the modified method of Yamashita et al [19], Pugh et al [12] and Kleinmann et al [6]. S. diastaticus YIY 345 transformants harboring the SGA gene were vigorously cultured in 1 liter of YPD medium at 30 o C for 4 days.…”

Section: Purification Of Sporulation-specific Glucoamylasementioning

confidence: 99%

“…The molecular weight was determined by gel filtration chromatography using HPLC column (Protein Pak 300 SW, Waters) equilibrated with 0.1 M sodium phosphate buffer (pH 6.8) [12]. A calibration curve was made with standard proteins (Sigma-Aldrich Chemical Co.); aldolase (M.W.…”

Section: Determination Of Molecular Weightmentioning

confidence: 99%

“…The mixtures were vortexed with glass-bead. The lysed cells were centrifuged and supernatant was recovered as the fraction of intracellular region [12].…”

Section: Protein Quantitationmentioning

confidence: 99%

“…The SGA is suggested to be glycosylated and located in the vacuole, the hydrolytic organelle of yeast which is analogous to mammalian lysosome. Thus, although it is detected in the intracellular region, the SGA contains the information for entry into the early stage of the secretory pathway of the cell [12].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Sporulation-Specific Glucoamylase of Saccharomyces diastaticus

2010

Journal of Life Science

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The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase I, II or III, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and 45 o C, respectively, whereas those for the extracellular glucoamylase were 5.0 and 50 o C. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

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Characterisation of Amylolytic Brewing Yeast

Vakeria¹,

Box²,

Bird

et al. 1996

Journal of the Institute of Brewing

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Amyioiytic brewing yeast can be used for the production of low carbohydrate beer and for maximizing fermentation efficiency. In this paper we describe the characterisation of amyioiytic brewing yeast in which the STA2 (DEX1) gene, which codes for an extracellular glucoamylase, was cloned under two different promoters; PGK (phosphoglycerate kinase) and GPD1 (sn-glycerol -3-phosphate dehydrogenase) present on episomal plasmids. Both amyioiytic strains were shown to ferment and degrade wort as efficiently as the control strain supplemented with an exogenous commercial glucoamylase, despite reduced intracellular glycogen levels (30% of wild-type). However, the nature of the promoter on the expression plasmid was shown to influence both the growth rate of the amyioiytic strains and the stability of the plasmids during non-selective growth. One of the strains containing plasmid pDVX4 {GPD promoter) was found to show high levels of stability when tested in ten successive pilot scale (8Hlitre) fermentations.

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Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae

Cited by 26 publications

References 62 publications

Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent Kinase Pho85p

Characterization of Sporulation-Specific Glucoamylase of Saccharomyces diastaticus

Characterisation of Amylolytic Brewing Yeast

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