Limits...
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.

Show MeSH

Related in: MedlinePlus

Altered expression of respiratory components and ROS scavengers by atg mutant cells.(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3045454&req=5

pone-0017412-g004: Altered expression of respiratory components and ROS scavengers by atg mutant cells.(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.

Mentions: It is known that the electron transport system is the major source of ROS production during respiratory growth [15], [17], [18], so we examined the accumulation of ROS during starvation in rho0 atg1Δ cells. Compared to atg1Δ cells (67.0% ROS accumulated cells), there was little accumulation of ROS in rho0 atg1Δ cells (3.0% of ROS accumulated cells) (Figure S4A and S4B), implying that respiration is the major source of ROS in atg mutants during nitrogen starvation. Alterations in components of the respiratory chain that affect reaction efficiency promote electron transport to oxygen and the generation of ROS [15]. To probe the composition of the respiratory pathway, we examined the expression of Cox4, a subunit of cytochrome C oxidase encoded in the nuclear genome, and Cox2, another cytochrome C oxidase component encoded in the mtDNA [19]. Expression of both Cox2 and Cox4 was increased in WT cells in response to nitrogen starvation, but no significant increase in Cox2 or Cox4 expression occurred in atg1Δ cells (Figure 4A). It is likely that ROS generation is more pronounced in atg mutants because respiratory chain function deviates from optimum conditions. On the other hand, the amount of Tim17, a subunit of the translocase of the mitochondrial inner membrane, slightly decreased in both WT and atg1Δ cells during starvation (Figure 4A), suggesting that mitochondrial protein composition changes during starvation and expression of these newly synthesized proteins depends on intact autophagy.


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

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

Altered expression of respiratory components and ROS scavengers by atg mutant cells.(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045454&req=5

pone-0017412-g004: Altered expression of respiratory components and ROS scavengers by atg mutant cells.(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.
Mentions: It is known that the electron transport system is the major source of ROS production during respiratory growth [15], [17], [18], so we examined the accumulation of ROS during starvation in rho0 atg1Δ cells. Compared to atg1Δ cells (67.0% ROS accumulated cells), there was little accumulation of ROS in rho0 atg1Δ cells (3.0% of ROS accumulated cells) (Figure S4A and S4B), implying that respiration is the major source of ROS in atg mutants during nitrogen starvation. Alterations in components of the respiratory chain that affect reaction efficiency promote electron transport to oxygen and the generation of ROS [15]. To probe the composition of the respiratory pathway, we examined the expression of Cox4, a subunit of cytochrome C oxidase encoded in the nuclear genome, and Cox2, another cytochrome C oxidase component encoded in the mtDNA [19]. Expression of both Cox2 and Cox4 was increased in WT cells in response to nitrogen starvation, but no significant increase in Cox2 or Cox4 expression occurred in atg1Δ cells (Figure 4A). It is likely that ROS generation is more pronounced in atg mutants because respiratory chain function deviates from optimum conditions. On the other hand, the amount of Tim17, a subunit of the translocase of the mitochondrial inner membrane, slightly decreased in both WT and atg1Δ cells during starvation (Figure 4A), suggesting that mitochondrial protein composition changes during starvation and expression of these newly synthesized proteins depends on intact autophagy.

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