V.N. Luzikov

ATP-dependent Proteolysis in Mitochondria m-AAA PROTEASE AND PIM1 PROTEASE EXERT OVERLAPPING SUBSTRATE SPECIFICITIES AND COOPERATE WITH THE mtHsp70 SYSTEM

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and them-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

Degradation and restoration of mitochondria upon deaeration and subsequent aeration of aerobically grown Saccharomyces Cerevisiae cells

Changes in the mitochondria of aerobically grown Saccharomyces cerevisiae cells upon deaeration and subsequent aeration of the medium were studied. 1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system. All the changes in mitochondria described occurred under conditions where cell division was insignificant. 2. Deaeration of the medium decreases the content of cytochromes b and aa3 in the mitochondrial fraction, cytochrome aa3 “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions. 3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure. 4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria. 5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole.

Quality control: from molecules to organelles

There is a vast body of literature on the quality control of protein folding and assembly into multisubunit complexes. Such control takes place everywhere in the cell. The correcting mechanisms involve cytosolic and organellar proteases; the result of such control is individual molecules with proper structure and individual complexes both with proper stoichiometry and proper structure. Obviously, the formation of organelles as such requires some additional criteria of correctness and some new mechanisms of their implementation. It is proposed in this article that the ability to carry out an integral (key) function may serve as a criterion of correct organelle assembly and that autophagy can be accepted as a mechanism eliminating the assembly mistakes.

From structure and functions of steroidogenic enzymes to new technologies of gene engineering

This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.

Studies on stabilization of the oxidative phosphorylation system. II. Electron transfer-dependent resistance of succinate oxidase and NADH oxidase systems of submitochondrial particles to proteinases and cobra venom phospholipase

Abstract Inactivation of the NADH oxidase system of submitochondrial particles by trypsin (or chymotrypsin) and inactivation of the succinate oxidase system by cobra venom phospholipase has been studied. The following results were obtained: 1. 1. Inactivation of NADH oxidase slows down in the presence of NADH and oxygen. The protective effect decreases or disappears completely when compounds hindering electron transfer (Zn2+, NAD+, deamino NAD+ and thionicotinamide NADH) are added to the incubation medium. The mentioned inhibitors per se do not increase the sensitivity of NADH oxidase to proteolytic enzymes. 2. 2. Similar results are obtained when deamino NADH is used as a substrate. In this case the protective effect also decreases in the presence of Zn2+ and deamino NAD+. 3. 3. The NADH analogues, unable to supply electrons for NADH oxidase, do not protect it from the action of proteinases. This refers to NADPH, thionicotinamide NADH, their oxidized forms, and also NAD+ and deamino NAD+. 4. 4. Succinate retards inactivation of the succinate oxidase by cobra venom phospholipase. Zn2+ and malonate interfere with electron transfer and reduce the protective effect of succinate. 5. 5. Succinate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) taken together under anaerobic conditions do not increase the resistance of succinate oxidase to the action of cobra venom phospholipase. Reduced TMPD in the presence of oxygen does not stabilize the system either. 6. 6. Inactivation of cytochrome oxidase in the submitochondrial particles by phospholipase is not retarded in the presence of the reduced TMPD and oxygen. Some stabilization of this fragment occurs in the presence of succinate. A conclusion is made that the rate of electron transfer is one of the factors determining the life-time of the respiratory chain in the presence of proteinases, as well as cobra venom phospholipase. Stabilization is possible only when electrons are transferred across all the labile sites of the multi-enzyme system.

Degradation of total cell protein at different stages ofSaccharomyces cerevisiae yeast growth

The fact that intracellular degradation of proteins is of great physiological importance is beyond doubt. Little is known, however, about the factors controlling protein catabolism. Interesting studies along this line have been carried out by Saheki and Holzer [ 1,2] , Lenney et al. [3] and by Cabib and Ulane [4]. As a result of these investigations, yeast intracellular proteinases and their inhibitors have been isolated and identified and their localization in the cell established. According to the data of Saheki and Holzer [2] , the activity of intracellular proteinases increases in the course of aerobic growth of S. cerevisiae. It was unknown, however, whether this increase was accompanied by a corresponding change in the rate of degradation of total cell protein of yeast. The present work fills this gap. It is demonstrated that during the exponential phase of growth as the activities of proteinases A and B increase one may observe increase in the rate of degradation of total cell protein of S. cerevisiae. It is shown that degradation of proteins in vivo can be suppressed by specific inhibitors of yeast proteinases.

Comparative study of thermal degradation of electron transfer particles and reconstituted respiratory chain. Relation of electron transfer to reactivation of submitochondrial particles

Abstract The thermal degradation of alkaline electron transfer particles and a respiratory chain reconstituted from simple enzyme complexes has been studied. It is shown that the reconstituted system loses its NADH oxidase activity rapidly and irreversibly at 38°. Electron transfer particles are also readily inactivated, but their NADH oxidase activity is restored completely after the addition of NADH under aerobic conditions. The NADH: cytochrome c oxidoreductase activity of submitochondrial particles undergoes similar changes, whereas the NADH: ferricyanide oxidoreductase and cytochrome oxidase activities do not change at all. Heating electron transfer particles results in a sharp drop in the steady-state levels of reduced cytochromes b, c(+c1), and aa3 at 6°, with NADH as the substrate. These results mean that one of the thermolabile electron transfer particles sites precedes cytochrome b in the respiratory chain. Preheated electron transfer particles are more sensitive to the action of chymotrypsin and potassium oleate than are the intact particles under the same conditions. Oleate prevents reactivation and causes substantial changes in the steady-state levels of reduced cytochromes in the preheated particles. Reactivation is accompanied by an increase in resistance of the particles to oleate and chymotrypsin. These observations suggest that the structure of electron transfer particles becomes slightly looser on heating, and is restored on reactivation. To reactivate the NADH oxidase system of submitochondrial particles NADH and oxygen are needed. Zn2+ inhibits electron transfer and thus prevents reactivation. Incubation of preheated particles with an excess of NADH and exogenous oxidized cytochrome c under anaerobic conditions does not result in complete recovery of NADH oxidase activity. Taking into account these results it must be concluded that reactivation is related to electron transfer through the entire NADH oxidase system, including cytochrome oxidase.

Formation and Functioning of Fused Cholesterol Side-Chain Cleavage Enzymes

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.

Effect of substrates on reconstitution of the mitochondrial respiratory chain under various conditions

Abstract The reconstitution of NADH oxidase, succinate oxidase and the complete respiratory chain from NADH: cytochrome c oxidoreductase, succinate: coenzyme Q oxidoreductase, cytochrome oxidase and cytochrome c was studied under various conditions. The formation of these multi-enzyme systems was prevented by cobra venom phospholipase. Reconstitution was possible in the presence of cobra venom only if the medium contained NADH (or succinate) and O2. Bovine serum albumin prevented the formation of NADH oxidase at low temperatures but hardly affected this process at 38–42°. It also increased the thermal stability of the reconstituted system. Reconstitution of NADH oxidase did not occur in the presence of potassium oleate, and bovine serum albumin completely eliminated the effect of the latter. However, bovine serum albumin did not protect the respiratory chain from the action of phospholipase. Therefore, the presence of NADH was necessary for the reconstitution of NADH oxidase at 38° in a medium containing bovine serum albumin and cobra venom. Thus, the natural agents indicated above have a substantial effect on the reconstitution of the respiratory chain. Reconstitution becomes possible with a strictly definite ratio between the effects of different external factors. A special part in the formation of the respiratory chain is played by substrates having a specific influence on its structure.

Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc–adrenodoxin reductase–adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.