When the cdc28 strain of Saccharomyces cerevisiae is incubated at restrictive temperatures, the yeasts digest themselves in 7 days by activating autophagic machinery. In parallel, the cell-proliferative activity decreases progressively after about 48 h. We have previously referred to this phenomenon as autophagic death. In the present study, we isolated and characterized a recessive mutant strain, dlp2, which delays the progression toward autophagic death. The cdc28 dlp2 cells contain many small vesicles instead of the large central vacuoles that are usually found in parental cdc28 cells. We showed that the dlp2 phenotype results from the presence of a single mutation in the gene ARL1 (ADPribosylation factor-like protein 1). Morphological and biochemical analyses of cdc28 dlp2 suggested that a defect in central vacuole formation is caused by aberrant membrane trafficking, although the protein-sorting to vacuoles is not affected. After a shift to a restrictive temperature, the components of the cytoplasm and nucleus of cdc28 dlp2 were condensed, with an accompanying formation of vesicles in the periphery (epiplasm) of the cells rather than an activation of the autophagic machinery. Introducing this ARL1 mutation into the normal ARL1 locus of the wild-type W303 strain again inhibited the progression of apoptotic cell death due to a defect in vacuole formation, which in this case was induced by the proapoptotic protein Bax. Thus, the ARL1 gene plays an important role in the formation of central vacuoles and in the progression of programmed cell death induced by cell-cycle arrest or Bax. These results suggested the presence of a programmed-cell death machinery in yeast that is similar to that related to the Type II cell death of mammalian cells characterized by autophagocytosis.

When the cdc28 strain of Saccharomyces cerevisiae is incubated at restrictive temperatures, the yeasts digest themselves in 7 days by activating autophagic machinery. In parallel, the cell-proliferative activity decreases progressively after about 48 h. We have previously referred to this phenomenon as autophagic death. In the present study, we isolated and characterized a recessive mutant strain, dlp2, which delays the progression toward autophagic death. The cdc28 dlp2 cells contain many small vesicles instead of the large central vacuoles that are usually found in parental cdc28 cells. We showed that the dlp2 phenotype results from the presence of a single mutation in the gene ARL1 (ADPribosylation factor-like protein 1). Morphological and biochemical analyses of cdc28 dlp2 suggested that a defect in central vacuole formation is caused by aberrant membrane trafficking, although the protein-sorting to vacuoles is not affected. After a shift to a restrictive temperature, the components of the cytoplasm and nucleus of cdc28 dlp2 were condensed, with an accompanying formation of vesicles in the periphery (epiplasm) of the cells rather than an activation of the autophagic machinery. Introducing this ARL1 mutation into the normal ARL1 locus of the wild-type W303 strain again inhibited the progression of apoptotic cell death due to a defect in vacuole formation, which in this case was induced by the proapoptotic protein Bax. Thus, the ARL1 gene plays an important role in the formation of central vacuoles and in the progression of programmed cell death induced by cell-cycle arrest or Bax. These results suggested the presence of a programmed-cell death machinery in yeast that is similar to that related to the Type II cell death of mammalian cells characterized by autophagocytosis.

We report herein that the level of reactive oxygen species (ROS) observed using dihydrorhodamine is much higher in either GTS1-deleted (gts1Delta) or GTS1-overexpressing (TMpGTS1) transformants than in the wild-type and that the levels of protein carbonyls are increased and the glutathione levels are decreased in both transformants. Consistently, the activities of superoxide dismutases (SODs) in both gts1Delta and TMpGTS1 were severely weakened, while the protein levels of both Cu/Zn-SOD and Mn-SOD were not so changed. As the intracellular copper levels were significantly increased in both transformants, we hypothesized that, in either gts1Delta or TMpGTS1 cells, the imbalanced homeostasis of copper induced an accumulation of ROS which caused inactivation of SODs further increasing ROS levels.