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Heterologous expression in yeast of mCYP11A1 fusions with different topogenic signals of yeast mitochondrial proteins for artificial channeling to different translocases of the inner membrane was used to gain insight in the mechanism of its topogenesis in mitochondria. To ensure insertion of the CYP11A1 domain into the inner mitochondrial membrane during the process of translocation, topogenic sequences containing transmembrane segments of Bcs1p(1-83), DLD(1-72), and full-sized AAC protein were used when constructing modified forms of CYP11A1, and the Su9(1-112) addressing signal was included to stimulate membrane insertion of CYP11A1 after its translocation to the matrix. Alternatively, to promote slippage of the hybrid molecules into the matrix, the hybrid of mCYP11A1 with the precursor of steroidogenic mitochondria matrix protein adrenodoxin (preAd) was designed. The extra sequences used for intramitochondrial sorting of CYP11A1 apparently ensured predicted topology of hybrid molecules in yeast mitochondria. All of the addressing sequences, containing transmembrane domains, provided effective insertion of the hybrid proteins AAC-mCYP11A1, Bcs1p(1-83)-mCYP11A1, DLD(1-72)-mCYP11A1 and Su9(1-116)-mCYP11A1 into the inner membrane. preAd-mCYP11A1 hybrid molecules were shown to be translocated across the inner membrane and tightly associated with the membrane on its matrix side but not membrane inserted. Measuring specific activities of hybrid proteins in the mitochondrial fractions upon addition of Ad and AdR showed that the hybrids predetermined for cotranslocational insertion of CYP11A1 into the inner membrane were more active in the reaction of cholesterol side-chain cleavage than those destined for insertion on the matrix side of the IM, the Ad-mCYP11A1 hybrid demonstrating only residual enzyme activity. The data obtained reinforce the proposal that complete transfer of the polypeptide chain into the matrix is not a necessary stage in its topogenesis, but rather persistent interaction of the polypeptide chain with the membrane during the process of translocation is of importance for heme binding, folding and membrane insertion.

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.

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins., (Copyright 1999 Academic Press.)

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 the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

An Escherichia coli strain providing hypersynthesis of a recombinant cytochrome P450scc precursor supplemented with the extra MetArgGlySerHis6GlyIleArg sequence at the NH2-terminus (6His-pP450scc) has been constructed. A procedure for isolation and purification of 6His-pP450scc from the cell homogenate has been elaborated. It has been found that the recombinant precursor is imported into isolated rat liver and rat heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing resulting in the mature size form of cytochrome P450scc. Modification of the targeting P450scc presequence resulting in its increased positive charge is supposed to relieve tissue-specific restrictions on the P450scc import into mitochondria.

It has been shown that pre-P-450scc of bovine adrenal cortex mitochondria synthesized in a rabbit reticulocyte lysate cell-free system, is translocated into isolated soybean cotyledon mitochondria, thereby taking the mature form size. This finding is suggestive of the occurrence of a specific receptor and maturase for pre-P-450scc in plant mitochondria. Thus, plant mitochondria can be used as recipients for the mammalian cholesterol hydroxylase system in an attempt to study the mechanism of its formation and preservation.

According to previous reports (Matocha M. F. and Waterman M. R. (1984) J. Biol. Chem. 259, 8672-8678 and (1986) Arch. Biochem. Biophys. 250, 456-460) import of the cytochrome P450 (scc) precursor into mitochondria is tissue-specific. The present paper shows that in vitro synthesized bovine cytochrome P450 (scc) precursor can be imported into isolated soybean cotyledon mitochondria and processed therein to the mature size product. This shows that heterologous import of the cytochrome P450 (scc) precursor is possible and that import into mitochondria of this precursor is not restricted to steroidogenic tissues.

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.

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.

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.

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]