calcimycin and ammonium-acetate

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

Topics: Acetates; Anti-Bacterial Agents; Autophagy; Calcimycin; Carbon; Chloramphenicol; Cycloheximide; Endopeptidases; Ethylmaleimide; Macrolides; Nitrogen; Proteins; Saccharomyces cerevisiae; Vacuoles

Guinea pig sperm were suspended in calcium-containing medium supplemented with various concentrations of the tetrasaccharide, stachyose. At concentrations up to and including 0.6 M, stachyose was without effect on the A23187-induced acrosome reaction. At 1.0 M stachyose, greater than 97% of sperm retained their acrosome after exposure to A23187, as judged by light microscopy. Electron microscopy demonstrated, however, that exocytotic membrane fusion had occurred, although with substantial retention of the acrosomal matrix. Sperm incubated in 1.0 M stachyose solutions also underwent exocytotic membrane fusion in the absence of A23187 and external calcium. Sperm suspended in 0.175 M ammonium chloride solution progressively lost motility over 30 min, but without acrosomal swelling. By contrast, sperm in 0.19 M ammonium acetate underwent substantial swelling of the acrosome within 2-5 min. 70-80% of these sperm were able to exclude the vital dye propidium iodide with their acrosomes swollen. These sperm underwent acrosomal shrinkage if resuspended in normal medium within 5-10 min, and the majority (60-70%) recovered some motility. These sperm could undergo an A23187-induced acrosome reaction. Electron microscopy indicated that swelling in ammonium acetate solution solubilizes much of the acrosomal matrix and causes internal fusion between adjacent regions of the outer acrosomal membrane. There was no exocytotic membrane fusion in ammonium acetate solution, however. The evidence suggests that there is no stachyose osmolality for guinea pig sperm which will suppress the membrane fusion associated with exocytosis, and that sufficiently high osmolalities cause exocytotic membrane fusion in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetates; Acrosome; Ammonium Chloride; Animals; Calcimycin; Cytoplasmic Granules; Exocytosis; Guinea Pigs; Intracellular Membranes; Male; Microscopy, Electron; Oligosaccharides; Osmolar Concentration; Spermatozoa