[9] Construction and use of gene fusions to lacZ (β-galactosidase) that are expressed in yeast

Rose, Mark D.; Botstein, David

doi:10.1016/0076-6879(83)01012-5

Cited by 346 publications

(209 citation statements)

References 11 publications

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“…Protein extraction, immunoblot, GFP-Atg8 processing, and alkaline phosphatase (Pho8Δ60) assays were performed as described previously (18,19,33). Yeast strains containing the β-galactosidase reporter plasmid ATG8p-LacZ(416) were grown in SMD or shifted to SD-N to induce autophagy and then examined with a β-galactosidase assay as described previously (34). ChIP was performed as described previously (35).…”

Section: Methodsmentioning

confidence: 99%

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Bartholomew

Suzuki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

123

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Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubuleassociated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.lysosome | membrane biogenesis | phagophore | stress | vacuole M acroautophagy, hereafter referred to as autophagy, is an evolutionarily conserved process used by eukaryotic cells for the bulk degradation of intracellular proteins and organelles (1). Autophagy is not only vital for cell survival in nutrient-poor conditions (2) but is also linked to various physiological processes, including immune defense, tumor suppression, and prevention of neurodegeneration (3). Whereas autophagy plays a primarily protective role, it can also contribute to cell death; thus, the magnitude of autophagy must be carefully regulated.Central to autophagy is the formation of autophagosomes (4), double-membrane-bound structures that engulf and deliver cytoplasmic materials to the vacuole/lysosome for degradation. During autophagosome formation, the autophagy-related ubiquitin-like protein Atg8/microtubule-associated protein 1 light chain 3 (LC3) covalently modifies phosphatidylethanolamine (PE). Almost one-fourth of the characterized autophagy-related (Atg) proteins in yeast are involved in the formation or stability of Atg8-PE, which plays a critical role in controlling expansion of the phagophore (the initial sequestering membrane), and in determining autophagosome size, thereby regulating autophagy activity (5, 6). Upon starvation, the level of ATG8 mRNA sharply increases, leading to a subsequent induction of the Atg8 protein level (7,8). The increase in the amount of Atg8 during autophagy is critical for supplying a sufficient amount of this protein to maintain normal levels of autophagy; yeast strains deficient in Atg8 induction generate abnormally small autophagosomes (5). Thus, characterization of how Atg8 protein levels are modulated is of tremendous importance both in understanding the regulation of autophagy and for the elucidation of potential therapeutic targets. However, little is known about the mechanisms regulating ATG8 transcription. ResultsUme6-Sin3-Rpd3 Complex Represse...

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Section: Methodsmentioning

confidence: 99%

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Bartholomew

Suzuki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

123

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“…PCR was performed with high fidelity Pfu polymerase (Stratagene). The constructs were verified by DNA sequencing (Genome Express S.A., Grenoble, France), prior to yeast transformations and ␤-galactosidase assays (31).…”

Section: Strain Construction Growth Conditions and Phenotypic Analymentioning

confidence: 99%

Genome-wide Analysis of the Response to Cell Wall Mutations in the Yeast Saccharomyces cerevisiae

Lagorce

Hauser

Labourdette

et al. 2003

Journal of Biological Chemistry

241

280

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Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity. To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e. fks1, kre6, mnn9, gas1, and knr4 mutants) that affects in a different manner the cell wall construction. Altogether, 300 responsive genes were kept based on high stringency criteria during data processing. Functional classification of these differentially expressed genes showed a substantial subset of induced genes involved in cell wall construction and an enrichment of metabolic, energy generation, and cell defense categories, whereas families of genes belonging to transcription, protein synthesis, and cellular growth were underrepresented. Clustering methods isolated a single group of ϳ80 up-regulated genes that could be considered as the stereotypical transcriptional response of the cell wall compensatory mechanism. The in silico analysis of the DNA upstream region of these co-regulated genes revealed pairwise combinations of DNA-binding sites for transcriptional factors implicated in stress and heat shock responses (Msn2/4p and Hsf1p) with Rlm1p and Swi4p, two PKC1-regulated transcription factors involved in the activation genes related to cell wall biogenesis and G 1 /S transition. Moreover, this computational analysis also uncovered the 6-bp 5 -AGCCTC-3 CDRE (calcineurindependent response element) motif in 40% of the coregulated genes. This motif was recently shown to be the DNA binding site for Crz1p, the major effector of calcineurin-regulated gene expression in yeast. Taken altogether, the data presented here lead to the conclusion that the cell wall compensatory mechanism, as triggered by cell wall mutations, integrates three major regulatory systems: namely the PKC1-SLT2 mitogen-activated protein kinase-signaling module, the "global stress" response mediated by Msn2/4p, and the Ca 2؉ /calcineurindependent pathway. The relative importance of these regulatory systems in the cell wall compensatory mechanism is discussed.Yeast and fungi are surrounded by a cell wall that is a complex structure essential for maintenance of the cell shape, prevention of lysis, and protection against harmful environmental conditions. The yeast cell wall architecture has been determined in detail over the past decade (1). It is a layered structure that is composed of ␤-1,3-and ␤-1,6-glucan (50 -60% of the cell wall dry mass), mannoproteins (40 -50%), and chitin (2%). ␤-1,3-Glucan and chitin form a fibrillar network to which mannoproteins are anchored, mostly through ␤-1,6-glucan. Some cell wall proteins, like the PIR family, can be directly linked to ␤-1,3-glucan (reviewed in Ref.2). The cell wall is not a rigid structure, since it endures all of the changes that the cell undergoes during division, morphogenesis, and differentiation.To ensure continuous integrity of the wall in accordance with its plasticity, complex mechanisms must be operating, which need to be strictly ...

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“…For the determination of ␤-galactosidase activity, transformants were assayed essentially as described previously (Rose and Botstein, 1983). Cells were grown to early exponential phase (A 600 ϭ 1) at 30°C before treatment with various stress conditions.…”

Section: ␤-Galactosidase Assaysmentioning

confidence: 99%

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Luikenhuis

Perrone

Dawes

et al. 1998

MBoC

218

217

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Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40 -52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using ␤-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. INTRODUCTIONGlutaredoxin from Escherichia coli was first discovered as a small, heat-stable protein required for the glutathione-dependent synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase (Holmgren, 1976). Glutaredoxin 1 is a 9-kDa protein that acts as a reduced glutathione (GSH)-dependent disulfide oxidoreductase by virtue of the two cysteine residues in its active site (Holmgren and Aslund, 1995). Later studies in mutants that lacked both glutaredoxin and thioredoxin revealed that E. coli actually contains three glutaredoxins (Grx1-3), with glutaredoxin 3 also able to function in ribonucleotide synthesis (Aslund et al., 1994). In contrast, glutaredoxin 2 was proposed to be the first member of a novel class of glutaredoxins that lack activity as hydrogen donors for ribonucleotide reductase (Vlamis-Gardikas et al., 1997). Glutaredoxins have subsequently been identified and isolated from various eukaryotes, including human, bovine, pig, yeast, and rice (Minakuchi et al., 1994;Holmgren and Aslund, 1995). The structure of these proteins has been highly conserved throughout evolution, particularly in the region of the active site (Wells et al., 1993;Holmgren and Aslund, 1995). However, despite extensive structural analysis, little is known regarding the biochemical function of these eukaryotic glutaredoxins in vivo.There appears to be considerable functional overlap between the glutaredoxin and thioredoxin systems. Similar to glutaredoxin, thioredoxin is a small, heatstable pro...

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[9] Construction and use of gene fusions to lacZ (β-galactosidase) that are expressed in yeast

Cited by 346 publications

References 11 publications

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Genome-wide Analysis of the Response to Cell Wall Mutations in the Yeast Saccharomyces cerevisiae

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

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