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Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

Suzuki SW, Onodera J, Ohsumi Y - PLoS ONE (2011)

Bottom Line: We found that buffering of the starvation medium dramatically restored the viability of atg mutants.Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA).We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions.

View Article: PubMed Central - PubMed

Affiliation: Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan.

ABSTRACT
Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

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Mitochondrial defect in atg mutants.(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B–C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
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pone-0017412-g003: Mitochondrial defect in atg mutants.(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B–C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.

Mentions: We next analyzed the mechanism by which autophagy-deficient cells lose their respiratory function. We examined if mitochondria retained mtDNA (mitochondria DNA) in atg mutant cells. Mitochondria were visualized by mitochondria targeted mCherry, and mtDNA was labeled with SYBR green. Both WT and atg1Δ cells contained mtDNA until 24 hours of nitrogen starvation (Figure S2). After 120 hours of starvation, the mitochondria in WT cells retained mtDNA, but nearly all of the atg1Δ cell lost their mtDNA (Figure 3A). We confirmed that the small colonies derived from culture grown in buffered starvation medium were comprised of rho0 cells, mitochondria DNA deficient cells, by DAPI staining (Figure S3), which stains both nuclear DNA and mtDNA. These results suggest that autophagy-defective mutants become rho0 cells during nitrogen starvation.


Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

Suzuki SW, Onodera J, Ohsumi Y - PLoS ONE (2011)

Mitochondrial defect in atg mutants.(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B–C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045454&req=5

pone-0017412-g003: Mitochondrial defect in atg mutants.(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B–C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Mentions: We next analyzed the mechanism by which autophagy-deficient cells lose their respiratory function. We examined if mitochondria retained mtDNA (mitochondria DNA) in atg mutant cells. Mitochondria were visualized by mitochondria targeted mCherry, and mtDNA was labeled with SYBR green. Both WT and atg1Δ cells contained mtDNA until 24 hours of nitrogen starvation (Figure S2). After 120 hours of starvation, the mitochondria in WT cells retained mtDNA, but nearly all of the atg1Δ cell lost their mtDNA (Figure 3A). We confirmed that the small colonies derived from culture grown in buffered starvation medium were comprised of rho0 cells, mitochondria DNA deficient cells, by DAPI staining (Figure S3), which stains both nuclear DNA and mtDNA. These results suggest that autophagy-defective mutants become rho0 cells during nitrogen starvation.

Bottom Line: We found that buffering of the starvation medium dramatically restored the viability of atg mutants.Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA).We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions.

View Article: PubMed Central - PubMed

Affiliation: Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan.

ABSTRACT
Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

Show MeSH
Related in: MedlinePlus