In this study, we showed links between impaired autophagy, mitochondrial injury and dysfunction, and, ultimately, cell death under conditions of nitrogen starvation.
We previously reported that autophagy-defective mutants cannot maintain the amino acid pool needed for protein synthesis, and this impairs the ability of cells to upregulate the expression of starvation-induced genes 
. In the present study, we found proteins involved in cellular respiration encoded both chromosomally (i.e. Cox4) and by the mtDNA (i.e. Cox2) upregulated under starvation conditions (). The ROS scavenger proteins Cta1 and Ctt1 also augmented under such conditions and autophagy is required for upregulation of these starvation-induced proteins (), consequently atg
mutants show the increased production of ROS and reduced ROS scavenging activity (), likely leading mitochondria dysfunction (). Consistently, treatment of cells with the antioxidant NAC suppressed the loss of respiratory activity and restored cell viability during starvation in atg
mutants ( and ). In this context, autophagy is essential for synthesis of minimal essential proteins to maintain mitochondrial function during nitrogen starvation.
Autophagy could contribute to the elimination of damaged mitochondria and, in this way, prevent the accumulation of ROS 
. Mitochondria are non-selectively incorporated into autophagosomes during starvation 
, but they are also selectively degraded via mitophagy, in which Atg32 is requisite 
. However, atg32
Δ cells showed nearly normal respiratory activity (), indicating that selective mitochondria elimination is not responsible for the observed respiratory defects caused in atg
mutants under starvation conditions. On the other hand, cells lacking ATG15
, which can form autophagosomes but not degrade the contents 
, exhibited respiratory deficient cells (). It is likely that sequestration of cytoplasmic components is not sufficient to retain respiratory function, but impaired autophagic degradation of cytoplasmic components is responsible for the maintenance of respiratory function.
In this study, we proposed that supply of amino acids via autophagy is important for the maintenance of respiratory function under starvation conditions. After 120 hours nitrogen starvation atg
mutants exhibit respiratory defects dramatically (WT: 3.8%, atg1
Δ: 75.5%) (). Recently, Zhang et al. presented that stationary phase atg
mutant cells shows higher spontaneous petite frequencies up to 6% than about 3% in WT cells 
. The total cellular amino acid levels in autophagy deficient cells drastically decrease under starvation conditions compared to those under nutrient rich conditions 
. It is likely that amino acid pool in nitrogen starved atg
mutant is smaller than that in stationary phase atg
mutant. It might account for the difference between rates of respiratory deficient cells of Zhang's result and our result.
The mechanisms by which excess ROS can induce the loss of mtDNA in autophagy-defective mutants remain unclear. It is known that the loss of mtDNA is caused by inhibition of mtDNA replication during cell division 
. Defect in mtDNA replication for several generations decreases inherited mtDNA number, and finally cells become rho0
mutants. However, nitrogen-starved cells are arrested at the first cell cycle 
, therefore a different unknown mechanism independent of cell proliferation must be involved.
We suggested that the maintenance of mitochondria function is important for cell survival during cell starvation. Recently, the relationship between autophagy and chronological life span had been discussed 
, and Fabrizio et al. presented that deletion of genes involved in autophagy or mitochondria functions shorten life span in Saccharomyces cerevisiae 
. Matecic et al. reported that raising medium pH to 6.0 also induces the extension of chronological life span in atg
. Thus, essential factors for long-term survival and adaptation to starvation conditions have common features, and neutralizing medium suppress short life span or starvation induced cell death in atg
mutants. Shorten chronological life span in autophagy-defective mutants may be at least partly attributed to mitochondria dysfunction.
We revealed that buffered starvation medium allows autophagy deficient cells to survive five days of nitrogen starvation (). It allows us to investigate autophagy during long nitrogen starvation. Until now, we observed that lipid droplets are transported to the vacuole in an autophagy dependent manner under prolonged starvation (manuscript in preparation). Thus, buffered starvation conditions will be useful to elucidate novel autophagic processes.
Autophagy has been implicated in a variety of essential cellular functions, and we have established an important role for autophagy in maintaining mitochondrial function by supporting essential protein synthesis, and mitochondria dysfunction is the major cause of starvation-induced cell death in autophagy-defective mutants. Further experiments in yeast will provide mechanistic insights into physiological roles of autophagy.