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Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae.

ABSTRACT: Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (-340 to -350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H(+)/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H(+)/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.

SUBMITTER: Ayer A

PROVIDER: S-EPMC3676407 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae.

Ayer Anita A Sanwald Julia J Pillay Bethany A BA Meyer Andreas J AJ Perrone Gabriel G GG Dawes Ian W IW

PloS one 20130607 6

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between ...[more]

PMID: 23762325

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Project description:BACKGROUND: Protein subcellular localization and differences in oxidation state between subcellular compartments are two well-studied features of the the cellular organization of S. cerevisiae (yeast). Theories about the origin of subcellular organization are assisted by computational models that can integrate data from observations of compositional and chemical properties of the system. PRESENTATION AND IMPLICATIONS OF THE HYPOTHESIS: I adopt the hypothesis that the state of yeast subcellular organization is in a local energy minimum. This hypothesis implies that equilibrium thermodynamic models can yield predictions about the interdependence between populations of proteins and their subcellular chemical environments. TESTING THE HYPOTHESIS: Three types of tests are proposed. First, there should be correlations between modeled and observed oxidation states for different compartments. Second, there should be a correspondence between the energy requirements of protein formation and the order the appearance of organelles during cellular development. Third, there should be correlations between the predicted and observed relative abundances of interacting proteins within compartments. RESULTS: The relative metastability fields of subcellular homologs of glutaredoxin and thioredoxin indicate a trend from less to more oxidizing as mitochondrion - cytoplasm - nucleus. Representing the overall amino acid compositions of proteins in 23 different compartments each with a single reference model protein suggests that the formation reactions for proteins in the vacuole (in relatively oxidizing conditions), ER and early Golgi (in relatively reducing conditions) are relatively highly favored, while that for the microtubule is the most costly. The relative abundances of model proteins for each compartment inferred from experimental data were found in some cases to correlate with the predicted abundances, and both positive and negative correlations were found for some assemblages of proteins in known complexes. CONCLUSION: The results of these calculations and tests suggest that a tendency toward a metastable energy minimum could underlie some organizational links between the the chemical thermodynamic properties of proteins and subcellular chemical environments. Future models of this kind will benefit from consideration of additional thermodynamic variables together with more detailed subcellular observations.

Project description:Cells cultured in a nutrient-limited environment can undergo adaptation, which confers improved fitness under long-term energy limitation. We have shown previously how a recombinant Saccharomyces cerevisiae strain, producing a heterologous insulin product, under glucose-limited conditions adapts over time at the average population level. Here, we investigated this adaptation at the single-cell level by application of fluorescence-activated cell sorting (FACS) and showed that the following three apparent phenotypes underlie the adaptive response observed at the bulk level: (i) cells that drastically reduced insulin production (23%), (ii) cells with reduced enzymatic capacity in central carbon metabolism (46%), and (iii) cells that exhibited pseudohyphal growth (31%). We speculate that the phenotypic heterogeneity is a result of different mechanisms to increase fitness. Cells with reduced insulin productivity have increased fitness by reducing the burden of the heterologous insulin production, and the populations with reduced enzymatic capacity of the central carbon metabolism and pseudohyphal growth have increased fitness toward the glucose-limited conditions. The results highlight the importance of considering population heterogeneity when studying adaptation and evolution. IMPORTANCE The yeast Saccharomyces cerevisiae is an attractive microbial host for industrial production and is used widely for manufacturing, e.g., pharmaceuticals. Chemostat cultivation mode is an efficient cultivation strategy for industrial production processes as it ensures a constant, well-controlled cultivation environment. Nevertheless, both the production of a heterologous product and the constant cultivation environment in the chemostat impose a selective pressure on the production organism, which may result in adaptation and loss of productivity. The exact mechanisms behind the observed adaptation and loss of performance are often unidentified. We used a recombinant S. cerevisiae strain producing heterologous insulin and investigated the adaptation occurring during chemostat growth at the single-cell level. We showed that three apparent phenotypes underlie the adaptive response observed at the bulk level in the chemostat. These findings highlight the importance of considering population heterogeneity when studying adaptation in industrial bioprocesses.

Dataset Information

Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae.

Publications

Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae.

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