[Polymorphism of membrane proteinases from mitochondria]. / Polimorfizm membrannyx proteinaz mitokhondrii.

Using native polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate, the detergent extracts of sonic submitochondrial particles (SMP) were separated into three protein fractions capable of accomplishing the proteolysis of cytochrome c and three other fractions catalyzing the hydrolysis of N-a-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-a-benzoyl-L-arginine-B-naphthylamide (BANA). The fractions isolated from the gel were subjected to a thorough anaylsis. Cytochrome c hydrolases were shown to have identical molecular weights (17000) but different isoelectric points (4.0, 4.2 and 4.4). The total cytochrome c hydrolase activity of these enzymes was inhibited by phenylmethylsulfonylfluoride but was insensitive to ethylenediaminetetraacetate and o-phenanthroline. Three BANA (BAPA) hydrolases have identical Mr values (approximately 17500) but different pI values (4.2, 4.3 and 4.7). Apart from the indicated hydrolases, the detergent extracts of SMP were shown to contain minor components with identical activities distinguished by the tightness of binding to the inner mitochondrial membrane, Mr and sensitivity to proteinase inhibitors. The observed phenomenon is considered to be due to the polymorphism of proteinases coupled with the inner mitochondrial membrane.

Physical state of the mitochondrial inner membrane as a factor controlling the proteolysis of mitochondrial translation products in yeast.

The degradation of mitochondrial translation products has been studied in Saccharomyces cerevisiae yeast. A high rate of degradation is observed in the early exponential phase of aerobic growth. Maturation of the yeast and glucose repression suppress the degradation. Anaerobic growth is also marked by a low breakdown rate of mitochondrial translation products. These variations did not correlate with the cytochrome c hydrolase activity of sonic submitochondrial particles (this activity was shown to reflect the general state of the proteolytic system of the inner mitochondrial membrane that is responsible for the breakdown of mitochondrially made polypeptides; see Novikova, L.A., et al. (1981) FEBS Lett. 135, 245-248). Experiments with lipid-soluble paramagnetic probes revealed significant variations in the physical state of the mitochondrial inner membrane, as judged from the comparison of the temperature-dependence plots of structural parameters obtained from the EPR spectra of the probes. The breakdown of mitochondrial translation products was, in general, the more rapid the lower was the temperature of the structural transition in the mitochondrial inner membrane and the higher was the relative content of unsaturated fatty acyl chains in the membrane phospholipids.

Detection, isolation and some properties of membrane proteinases from yeast mitochondria.

Three cytochrome c hydrolase species were found in the 0.05% SDS extract from submitochondrial particles. Their polypeptides all have a molecular weight of 17 000 but differ in pI values (4.0, 4.2 and 4.4), as shown by SDS-polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The activity of pooled cytochrome c hydrolases is sensitive to PMSF, pCMPS , and leupeptin but insensitive to EDTA or o-phenanthroline. Besides the cytochrome c-hydrolyzing enzymes, the SDS extract contains three protein components with BAPA ( BANA )-hydrolyzing activity, which also show similar molecular weights (17 5000) but different pI values (4.2, 4.3 and 4.7). It is supposed that at least some of the enzymes mentioned are involved in the intramembrane proteolysis of polypeptides synthesized on mitoribosomes .

Correlation between the rate of proteolysis of mitochondrial translation products and fluidity of the mitochondrial inner membrane in Saccharomyces cerevisiae yeast. Alteration of the rate of proteolysis under glucose repression.

Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids.

[Mitochondrial proteinases of the yeast Saccharomyces cerevisiae: electrophoretic detection, substrate specificity and sensitivity to inhibitors]. / Mitokhondrial'nye proteinazy drozhzhei Saccharomyces cerevisiae: élektroforeticheskoe vyiavlenie, substratnaia spetsifichnost' i chuvstvitel'nost' k ingibitoram.

The proteins of submitochondrial particles solubilized with 0.1% Triton X-100 were separated by polyacrylamide gel electrophoresis. Hydrolysis of several proteinase substrates was registered directly in the gel after completion of electrophoresis. According to the data obtained the inner mitochondrial membrane contains one or two enzymes which catalyze hydrolysis of cytochrome c as well as one or two enzymes splitting synthetic substrate of trypsin-like proteinases, e. g. N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-alpha-benzoyl-L-arginine-beta-naphthylamide (BANA). Submitochondrial particles were shown to catalyze hydrolysis of 3H-labelled cytochrome c. This activity is suppressed by the same inhibitors as the hydrolysis of mitochondrial translation products, i. e. phenyl-methylsulfonylfluoride, p-chloromercuribenzosulfonate, leupeptin and antipain. Presumably these two processes are catalyzed by the same enzyme localized in the inner mitochondrial membrane. Physiological functions of BAPA- and BANA-hydrolyzing enzyme(s) are still unclear.

[Changes in proteinase A and B activity during glucose repression in the yeast Saccharomyces cerevisiae]. / Izmenenie aktivnostei proteinaz A i B pri gliukoznoi repressii drozhzei Saccharomyces cerevisiae.

The divisible and indivisible yeast S. cerevisiae were subjected to glucose repression by increasing the glucose content in the growth medium up to 10%. Under these conditions the total activities of both proteinases did not change significantly, while those of free (i.e. not bound to natural inhibitors) proteinases were increased manyfold. This effect is probably due to liberation of the proteinases from the vacuoles and digestion of cytosolic proteinase inhibitors.

[Mitochondrial proteinase of yeast splitting the products of mitochondrial translation]. / Mitokhondrial'naia proteinaza drozhzhei, osushchestvliaiashchaia rasshcheplenie produktov mitokhondrial'noitransliatsii.

It has been shown that mitochondria of the yeast Saccharomyces cerevisiae contain proteinase, which is bound to the inner mitochondrial membrane and catalyzes the hydrolysis of mitochondrial translation products in vitro. The efficiency of proteolysis depends on the state of mitochondria: e.g. the degradation of completely formed organelles corresponding to stationary cells, is twice as low as compared to the "young" organelles typical for the beginning of a logarithmic phase of growth. The proteolysis of mitochondrial translation products can occur not only in mitochondria, but also in "inside out" submitochondrial particles. In order to prove the absence of concomitant vacuolar proteinases in preparations of mitochondria and submitochondrial particles, the specific antisera against proteinases A and B have been used. The activity of mitochondrial proteinase is completely inhibited by the natural peptide inhibitors antipain and chymostatin. Of special importance is the fact that another natural peptide inhibitor--leupeptin, having no effect on the activities of vacuolar proteinases, significantly decreases the rate of hydrolysis of mitochondrial translation products. The role of yeast mitochondrial proteinase in regulation of mitochondrial formation is discussed.

Glucose repression and autolysis of Saccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase.

Alterations in the localization of acid phosphatase in Saccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondrial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm, whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm. No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.

[Cytochemical and biochemical determination of acid phosphatase activity in yeasts]. / Tsitokhimicheskoe i biokhimicheskoe opredelenie aktivnosti kisloi fosfatazy v drozhzhakh.

Optimal conditions of the cytochemical assay for acid phosphatase in protoplasts and whole cells of S. cerevisiae have been described. Dimethyl sulfoxide was used to increase the permeability of the yeast cell envelope. In the yeast cells, grown up to the end of the exponential phase, acid phosphatase is shown to be located mainly in the central vacuole and on the cell envelope surface. A considerable activity of acid phosphatase is demonstrable on the surface of the plasma membrane and within adjacent vesicles that represent, presumably, part of the endoplasmic reticulum. Acid phosphatase can be considered as a marker enzyme for yeast cell vacuoles.

Proteolysis of the products of mitochondrial protein synthesis in yeast mitochondria and submitochondrial particles.

Degradation of mitochondrial translation products in Saccharomyces cerevisiae mitochondria was studied by selectively labelling these entities in vivo in the presence of cycloheximide and following their fate in isolated mitochondria. One-third to one-half of the mitochondrial translation products are shown to be degraded, depending on the culture growth phase, with an approximate half-life of 35 min. This process is shown to be ATP-dependent, enhanced in the presence of puromycin and inhibited by chloramphenicol. Further, the proteolysis is suppressed by detergents and is insensitive to antisera against yeast proteinases A and B when measured in mitochondria or 'inside-out' submitochondrial particles. It is concluded that the breakdown of mitochondrial translation products is most probably due to the action of endogenous proteinase(s) associated with the mitochondrial inner membrane. This proteinase is inhibited by phenylmethanesulphonyl fluoride, leupeptin, antipain and chymostatin.

A cytochemical study of the localization of acid phosphatase in Saccharomyces cerevisiae at different growth phases.

The localization of acid phosphatase in the yeast Saccharomyces cerevisiae at different growth phases had been studied. It was shown to be crucial for authentic location of acid phosphatase that the cytochemical reaction be performed on whole cells. Dimethylsulphoxide was used to alleviate the effects of fixation of the yeast cells with glutaraldehyde; the sulphoxide did not affect the distribution of acid phosphatase in the cells. It has been established that in exponentially-growing cells acid phosphatase is localized mostly in small vacuolar compartments. In mature cells, the bulk of acid phosphatase is found in the central vacuole, although a significant amount of the enzyme is detectable in the plasma membrane and the adjacent vesicles.

The lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae. A novel method for protein half-life determination.

A method for the determination of the half-life of mitochondrial translation products in yeast in vivo is proposed. The method uses inhibitors of cytoplasmic and mitochondrial protein synthesis and is based on double-labelling pulse-chase techniques, the second label being used to estimate 'post-incorporation' during the 'chase'. For the first time the difference between post-incroporation and the widely known recycling of the label is considered. These studies show that, in the turnover of mitochondrial translation products, the problem is of post-incorporation into mitochondria (especially from the cell sap) is predominant. The results obtained with this procedure indicate that the half-life of the products of mitochondrial protein synthesis in yeast at the late-exponential phase is about 60 min. The results suggest that mitochondrial transplantation products are subject to proteolysis to acid-soluble forms.

[Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts]. / Proteoliz produktov mitokhondrial'nogo belkovogo sinteza v izolirovannykh mitokhondriiakh drozhzhei Saccharomyces cerevisiae.

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.

[Lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae yeasts]. / Labil'nost' produktov mitokhondrial'nogo belkovogo sinteza v drozhzhakh Saccharomyces cerevisiae

Estimation of the rate of degradation of the products of mitochondrial protein synthesis in S. cerevisiae cells is reported. The method developed for this purpose is based on pulse incorporation of a labeled amino acid in the presence of an inhibitor of cytoplasmic protein synthesis and allows one to monitor postincorporation of the label. The label incorporated is shown to be rapidly released from mitochondria. Its content is decreased 2-fold during 20-30 min at the beginning and 50-60 min at the end of the exponential phase of growth. The label is detected in cytosol proteins and the TCA-soluble fraction of mitochondria, which is indicative of possible proteolysis of mitochondrial membrane proteins. Since release of the label does not undergo inhibition by specific inhibitors of yeast cell proteinases (pepstatin and phenylmethylsulfonyl fluoride), it may be assumed that these proteinases are either not involved in the digestion of the products of mitochondrial protein synthesis or do not represent a rate-limiting step of the process.

[Degradation of intracellular proteins at different stages of growth of Saccharomyces cerevisiae]. / Degradatsiia vnutrikletochnykh belkov na raznykh fazakh rosta drozhzhei Saccharomyces cerevisiae.

The rate of degradation of intracellular proteins at different growth stages of Saccharomyces cerevisiae yeast was determined. It has been demonstrated that the rate of degradation of intracellular proteins increases 2--3-fold at the late exponential phase. The increase was accompanied by corresponding changes in the activities of yeast proteinases A and B. In the presence of specific yeast proteinase inhibitors (pepstatin and phenylmethylsulfonyl fluoride) the rate of protein degradation in vivo decreased. The intermediate products of cell protein degradation have been found. These TCA-insoluble products could be extracted by various solvent systems. Their subsequent brakdown was suppressed by specific proteinase inhibitors.