For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.

Reduction in klotho gene expression causes accelerated senescence in klotho mutant mice. We have now found two key substances, phosphorus and zinc, which affect the appearance of klotho phenotypes. Klotho mutant homozygotes fed nonpurified diet with a phosphorus concentration of 1.03 g/100 g showed typical klotho phenotypes. However, most of the klotho phenotypes no longer appeared in male homozygotes fed a 0.4 g/100 g phosphorus diet. These homozygotes were capable of spermatogenesis. In the kidneys of the rescued male homozygotes, klotho protein expression was clearly detected. On the other hand, female klotho mice required supplementation of 0.25 g/100 g zinc orotate to the 0.4 g/100 g phosphorus diet to be rescued. Unlike in the rescued male mice, klotho protein levels in the kidneys of the rescued females were quite low. Wild-type (C3H/He) mice fed 1.5 or 1.0 g/100 g phosphorus diets had lower klotho protein expression in the kidneys than those fed a 0.4 g/100 g phosphorus diet (Kruskal-Wallis test, P < 0.05). These results indicate that dietary phosphorus and zinc modulate the phenotypes of klotho mice, and that klotho expression in the kidneys is regulated not only in klotho mutant mice, but also in wild-type mice.

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