Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y
Department of Electrical Engineering, Kogakuin University, Tokyo, Japan.
J Cell Biol. 1992 Oct;119(2):301-11. doi: 10.1083/jcb.119.2.301.
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.
为了确定液泡蛋白水解在酿酒酵母中的生理作用和机制，将缺乏蛋白酶A、B和羧肽酶Y的突变细胞从营养培养基转移至缺乏各种营养成分的合成培养基中，并研究其液泡的形态变化。在营养缺乏的培养基中孵育1小时后，液泡中出现了一些球体，并通过布朗运动活跃移动。这些球体数量逐渐增加，3小时后几乎完全充满液泡。在它们积累的过程中，液泡区室的体积也增加了。电子显微镜检查显示，这些球体被一层单位膜包围，该单位膜看起来比任何其他细胞内膜都薄。球体的内容物在形态上与细胞质难以区分；这些球体含有细胞质核糖体、糙面内质网、线粒体、脂质颗粒和糖原颗粒，球体中细胞质核糖体的密度与细胞质中核糖体的密度几乎相同。球体的直径在400到900纳米之间。通过对大隅和荒九（大隅洋，荒九洋。1981。《生物化学杂志》256:2079 - 2082）方法的改进，制备了积累了这些球体的液泡。分离得到的液泡含有核糖体，并显示出细胞质酶葡萄糖 - 6 - 磷酸脱氢酶的潜在活性。这些结果表明，这些球体将细胞质隔离在了液泡中。我们将这些球体命名为“自噬体”。液泡中自噬体的积累不仅由氮饥饿诱导，还由碳和单一氨基酸等营养物质的耗尽诱导，这些营养物质的耗尽会导致细胞周期停止。遗传分析表明，液泡中自噬体的积累是由于缺乏PRB1产物蛋白酶B的结果，PRB1基因的破坏证实了这一结果。在苯甲基磺酰氟（PMSF）存在的情况下，野生型细胞在营养缺乏条件下，与多种蛋白酶缺陷型突变体或PRB1基因被破坏的细胞一样，在液泡中积累自噬体。由于从正常细胞培养物中去除PMSF后自噬体迅速消失，它们一定是正常自噬过程中的一个中间产物。这是关于营养缺乏条件诱导酵母细胞液泡中细胞质成分广泛自噬降解的首次报道。
