A rapid permeabilization procedure for accurate quantitative determination of β-galactosidase activity in yeast cells

Kippert, Fred

doi:10.1016/0378-1097(95)00113-j

Cited by 51 publications

(53 citation statements)

References 10 publications

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“…In one set, stressors were added and kept for stipulated period as given. To activate the HOG pathway, 0.5 M NaCl for 1 h; to activate the CWI pathway, 3 mM caffeine for 4 h; to stimulate the UPR, 2 mM DTT for 4 h; to induce the pheromone signaling pathway, 5 μM α-factor for 2 h. Cells were finally harvested and subjected to rapid permeabilization procedure as described earlier (Kippert, 1995). Activity is reported as percentage of Miller units.…”

Section: Methodsmentioning

confidence: 99%

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

Bankapalli

Mishra

Raychaudhuri

2017

Front. Cell. Infect. Microbiol.

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VopE, a mitochondrial targeting T3SS effector protein of Vibrio cholerae, perturbs innate immunity by modulating mitochondrial dynamics. In the current study, ectopic expression of VopE was found to be toxic in a yeast model system and toxicity was further aggravated in the presence of various stressors. Interestingly, a VopE variant lacking predicted mitochondrial targeting sequence (MTS) also exhibited partial lethality in the yeast system. With the aid of yeast genetic tools and different stressors, we have demonstrated that VopE and its derivative VopEΔMTS modulate cell wall integrity (CWI-MAPK) signaling pathway and have identified several critical residues contributing to the lethality of VopE. Furthermore, co-expression of two effectors VopEΔMTS and VopX, interfering with the CWI-MAPK cellular pathway can partially suppress the VopX mediated yeast growth inhibition. Taken together, these results suggest that VopE alters signaling through the CWI-MAPK pathway, and demonstrates the usefulness of yeast model system to gain additional insights on the functionality of VopE.

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Section: Methodsmentioning

confidence: 99%

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

Bankapalli

Mishra

Raychaudhuri

2017

Front. Cell. Infect. Microbiol.

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“…The reaction was stopped by adding 400 µl 1.5 M Na 2 CO 3 . After centrifugation for 1 min, supernatants were taken and their absorbance assayed at 420 nm [42]. The obtained absorbance values were normalized according to the OD values of cells.…”

Section: Methodsmentioning

confidence: 99%

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

et al. 2011

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Boron is an essential micronutrient for plants, and it is beneficial for animals. However, at high concentrations boron is toxic to cells although the mechanism of this toxicity is not known. Atr1 has recently been identified as a boron efflux pump whose expression is upregulated in response to boron treatment. Here, we found that the expression of ATR1 is associated with expression of genes involved in amino acid biosynthesis. These mechanisms are strictly controlled by the transcription factor Gcn4 in response to boron treatment. Further analyses have shown that boron impaired protein synthesis by promoting phosphorylation of eIF2α in a Gcn2 kinase dependent manner. The uncharged tRNA binding domain (HisRS) of Gcn2 is necessary for the phosphorylation of eIF2α in the presence of boron. We postulate that boron exerts its toxic effect through activation of the general amino acid control system and inhibition of protein synthesis. Since the general amino acid control pathway is conserved among eukaryotes, this mechanism of boron toxicity may be of general importance.

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“…β‐galactosidase activities were measured on whole cells by the method of Kippert (1995). The transformed strains were grown in different media supplemented with galacturonic acid (Sigma), digalacturonic acid (Sigma) or polygalacturonic acid (FLUKA, pectic acids from apple; degree of esterification <5%) at different concentrations (see text).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

Gognies¹,

Simon²,

Belarbi³

2001

Yeast

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Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-speci®c polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been ampli®ed and used to construct gene fusions with the b-galactosidase gene. On the basis of b-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and ®lament formation conditions.

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A rapid permeabilization procedure for accurate quantitative determination of β-galactosidase activity in yeast cells

Cited by 51 publications

References 10 publications

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

VopE, a Vibrio cholerae Type III Effector, Attenuates the Activation of CWI-MAPK Pathway in Yeast Model System

Boron Stress Activates the General Amino Acid Control Mechanism and Inhibits Protein Synthesis

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

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