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.

/pdf/autophagy-in-yeast-demonstrated-with-proteinase-deficient-z3zmckzuho.pdf

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

The fungal vacuole: composition, function, and biogenesis.

Fifty-nine mutants with reduced ability to cleave the chymotrypsin substrate N-acetyl-DL-phenylalanine beta-naphthyl ester have been isolated in S. cerevisiae. All have reduced levels of one or more of the three well-characterized proteinases in yeast. All have reduced levels of proteinase C (carboxy-peptidase Y). These mutations define 16 complementation groups.

https://www.genetics.org/content/genetics/85/1/23.full.pdf

Proteinase mutants of Saccharomyces cerevisiae.

Publisher Summary This chapter describes the conditions and procedures that affect the levels and activities of the vacuolar proteases and present both genetic and biochemical assaying methods for coping with protease problems. Protease B activity in colonies can be assayed using an overlay test. It is based on the principle that states that protease B, which is freed from cells by lysis and from its inhibitor by the SDS present in the overlay, solubilizes the particles of Hide Powder Azure in the overlay, uncovering the colony and surrounding it with a clear halo. Mutant colonies remain covered. Carboxypeptidase Y activity in colonies can be assessed by using an overlay test that relies on the esterolytic activity of the enzyme. The substrate is N-acetyl-DL-phenylalanine β-naphthyl ester, cleavage of which, in colonies anyway, is catalyzed only by CpY. In biochemical analyses, commonly used assay for protease A activity measures the release of tyrosine-containing acid-soluble peptides from aciddenatured hemoglobin. Protease A is apparently the only protease to catalyze the reaction at acid pH. It is reported the use of a protease-deficient strain can ease protease problems. Strains bearing the pleiotropic pep4-3 mutation have greatly reduced, but not zero, levels of PrA, PrB CpY, and ApI. Strains such as EJ101 that carry prb1-1122 have also been used successfully in some purifications. Use of a double mutant is recommended that carries mutations both in the PEP4 gene and the PRB1 gene.

Tackling the protease problem in Saccharomyces cerevisiae.

Isoelectric points and molecular weights of proteins.

Proteolytic activities in yeast.

The partially purified tryptophan synthase inactivating enzymes I and II were compared with the earlier described proteinases A, B and C by studying their catalytic properties, their sensitivity against a serine proteinase inhibitor and their temperature stability.



1
Tryptophan synthase inactivase activities of inactivase I and II coincided with hemoglobin hydrolyzing and azocoll hydrolyzing activities, respectively, on hydroxyapatite column chromatography.

2
Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were insensitive to the treatment with phenylmethylsulfonyl fluoride, whereas inactivase and azocoll hydrolyzing activities of the inactivase II fraction were almost completely inhibited by phenylmethylsulfonyl fluoride.

3
The N-acetyltyrosine ethyl ester (Ac-Tyr-OEt) hydrolyzing activity of an inactivase I fraction from an hydroxyapatite column could be separated from the inactivase activity by DEAE-Sephadex column chromatography. Because of its esterolytic properties, the separated fraction corresponds to proteinase C.

4
Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were stable at pH 6.0 and 37°C, but unstable at pH 8.0 and 37°C, while inactivase and azocoll hydrolyzing activities of the inactivase II fraction were unstable even at pH 6.0 and 37°C.





From these studies it is concluded that inactivase I is identical to proteinase A, inactivase II is identical or very similar to proteinase B and that proteinase C had no tryptophan synthase inactivase activity.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03377.x

Comparisons of the Tryptophan Synthase Inactivating Enzymes with Proteinases from Yeast

Inhibitors for yeast proteinase A have been purified from boiled extracts from baker's yeast. Upon SP-Sephadex C-25 column chromatography, four different peaks of proteinase A inhibitory activity appear. The inhibitors IA2 and IA3 have been further purified by QAE-Sephadex chromatography and preparative disc electrophoresis. Both inhibitors are homogeneous in dodecylsulfate-gel electrophoresis. From the electrophoretic mobilities in dodecylsulfate-polyacrylamide gels, a molecular weight of 6100 ± 200 was estimated for both inhibitors. In the absence of dodecylsulfate, both inhibitors showed one predominant band and a second faint, diffuse band. As a result of Sephadex G-75 gel filtration, a molecular weight of 23000 ± 2000 was obtained for both inhibitors, indicating an aggregation of the monomers to a tetramer. Upon electrofocusing in acrylamide gels the isoelectric points of the inhibitors were found to be 5.7 for IA2 and 6.3 for IA3. Both inhibitors inhibited proteinase A. Neither proteinases B and C from yeast nor pepsin were inhibited. Both inhibitors were inactivated after incubation with purified yeast proteinase B, but not with the yeast proteinases A or C. The inhibition of proteinase A at high inhibitor/proteinase ratios was more pronounced at pH 5 and 7 as compared to pH 3. An autocatalytic activation of a mixture of inhibited proteinases A, B and C by addition or liberation of traces of active proteinase A or B is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03697.x

Purification and Characterization of Macromolecular Inhibitors of Proteinase A from Yeast

Sephadex G-100 gel filtration of a purified mixture of tryptophan-synthase-inactivating enzymes I and II from Saccharomyces cerevisiae, which had previously been identified as yeast proteinases A and B, yields two coincident peaks of tryptophan-synthase-inactivating activity and proteinase A and B activities. The peaks correspond to molecular weights of approximately 100 000 and 50 000. It could be demonstrated, with purified proteinases A and B, that the high molecular weight peaks is due to the formation of a stoichiometric complex between proteinases A and B. The AB complex can easily be separated into its constituent enzymes by DEAE-Sephadex chromatography. When in the AB complex, proteinase B molecules still bind the specific proteinase B inhibitor from yeast.

Takeyori Saheki

Papers

Proteolytic activities in yeast.

Comparisons of the Tryptophan Synthase Inactivating Enzymes with Proteinases from Yeast

Purification and Characterization of Macromolecular Inhibitors of Proteinase A from Yeast

Formation of a Complex between Yeast Proteinases A and B