Principles of control over formation of structures responsible for respiratory functions of mitochondria.

Topogenesis of mitochondrial proteins includes their synthesis in cytosol and mitochondria, their translocation across the outer and inner membranes, sorting to various mitochondrial compartments, and assembly of different protein complexes. These complexes are involved in transport functions, electron transfer through the respiratory chain, generation of transmembrane electrochemical potential, oxidative phosphorylation of ADP into ATP, etc. To perform these functions, a special stringent control is required over formation of submitochondrial structures and the mitochondrion as a whole. Such control is expected to rigorously eliminate not only misfolded proteins but also incorrectly incorporated subunits and is realized in mitochondria by means of numerous proteases with different functions and localizations. In the case of more complicated protein formations, e.g. supercomplexes, the protein quality is assessed by their ability to realize the integral function of the respiratory chain and, thus, ensure the stability of the whole system. Considering supercomplexes of the mitochondrial respiratory chain, the present review clearly demonstrates that this control is realized by means of various (mainly vacuolar) proteases with different functions and localizations. The contemporary experimental data also confirm the author's original idea that the general mechanism of assembly of subcellular structures is based on the "selection by performance criterion" and "stabilization by functioning".

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

Abstract (3/4) 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.

Import of hybrid forms of CYP11A1 into yeast mitochondria.

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.

Comparative study of topogenesis of cytochrome P450scc (CYP11A1) and its hybrids with adrenodoxin expressed in Escherichia coli cells.

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2- or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH2-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.

Expression of cytochrome P450scc in Escherichia coli cells: intracellular location and interaction with bacterial redox proteins.

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.

Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells.

Using the pTrc99A/P450scc vector, a plasmid was constructed in which cDNAs for cytochrome P450scc, adrenodoxin reductase, and adrenodoxin are situated in a single expression cassette. This plasmid was shown to direct the synthesis of all the above proteins in Escherichia coli. Their localization in the E. coli cells and stoichiometry were determined. Cell homogenates exhibited cholesterol hydroxylase/lyase activity, due to catalytically active forms of all three proteins. Thus, the full set of constituents of the mammalian cholesterol hydroxylase/lyase system was shown to be synthesized in bacterial cells for the first time.

Use of the Addressing Sequence of Yeast D-Lactate Dehydrogenase for Insertion of CYP11A1p into the Inner Membrane of Yeast Mitochondria.

Mammalian cytochrome P450scc (CYP11A1p) is a pseudointegral protein of the inner membrane of mitochondria with the active center exposed in the matrix. Upon import of the CYP11A1p precursor into yeast mitochondria, only a minor part was incorporated into the inner mitochondrial membrane and acquired catalytic activity (Kovaleva, I. E., Novikova, L. A., Nazarov, P. A., Grivennikov, S. I., and Luzikov, V. N. (2003) Eur. J. Biochem., 270, 222-229). The present work is an attempt to increase the efficiency of this process by substitution of the inherent N-terminal presequence of CYP11A1p by the addressing signal of D-lactate dehydrogenase (D-LD) of the yeast Saccharomyces cerevisiae. D-LD is known to be inserted into the inner membrane of mitochondria through its transmembrane domain located close to the N-terminus of the polypeptide chain in such a way that the protein globule is exposed in the intermembrane space. The hybrid protein D-LD(1-72)-mCYP11A1p synthesized in yeast cells was imported into yeast mitochondria, underwent processing, and was inserted into the inner membrane on the side of the intermembrane space. In the presence of adrenodoxin and adrenodoxin reductase, the hybrid protein exhibited cholesterol side-chain cleavage activity. Thus, CYP11A1p insertion into the inner membrane of mitochondria mediated by the D-LD topogenic signal resulted in the catalytically active mCYP11A1p domain in the hybrid protein.

Quality control: proteins and organelles.

This review summarizes materials on the mechanisms of intracellular degradation of proteins whose topogenesis is disturbed at one stage or another. Chaperone and proteolytic systems involved in this process in the endoplasmic reticulum, mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct subcellular compartments of procaryotic cells are considered. The available data suggest that living cells contain numerous systems keeping under control both folding of newly synthesized and newly imported polypeptide chains and their incorporation into heterooligomeric complexes. The point of view is elaborated that organelle formation is controlled not only at the level of individual protein molecules but also at the supermolecular level when whole organelles incapable of carrying out their integral key functions become targets for partial or total elimination. This type of control is realized through an autophagic mechanism involving lysosomes/vacuoles.

On the effect of cholesterol on the fate of CYP11A1 imported into yeast mitochondria in vivo.

It has earlier been shown that CYP11A1 (cytochrome P450scc precursor), synthesized in yeast cells, is imported into yeast mitochondria. However, in large part the foreign protein undergoes degradation or aggregates. In this work, we tried to prevent aggregation of CYP11A1 and stimulate its insertion into the mitochondrial inner membrane by substituting cholesterol (a substrate for cytochrome P450scc) for ergosterol in yeast cells. To this end, an ergosterol-deficient Saccharomyces cerevisiae mutant, growing in the presence of cholesterol and expressing a modified bovine CYP11A1 gene, was used. Under defined conditions, the mitochondrial respiratory system developed in this yeast and CYP11A1 with the CoxIV targeting presequence was imported into the mitochondria, being then proteolytically processed. However, substitution of cholesterol for ergosterol did not result in lowered aggregation of the imported CYP11A1 and its increased content in the SMP fraction. Hence, the presence of cholesterol is not instrumental in proper intramitochondrial compartmentalization and folding of CYP11A1.

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.

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.

Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

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.

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

Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast.

A plasmid for effective expression of recombinant DNA encoding a hybrid protein composed of the N-terminal targeting presequence of subunit IV of yeast cytochrome c oxidase preceding the mature polypeptide chain of bovine cytochrome P450scc (pCoxIV-CYP11A1) in yeast has been constructed. It has been shown that this protein, when synthesized in yeast cells, in imported into mitochondria and undergoes proteolytic processing, thus yielding a product of molecular mass corresponding to that of mature cytochrome P450scc. However, only insignificant portion of the imported protein proves to be inserted into the inner membrane of heterologous mitochondria. The membrane-bound cytochrome P450scc exhibits cholesterol hydroxylase activity towards 22R-hydroxycholesterol in the presence of exogenous adrenodoxin and adrenodoxin reductase. This fact indicates that the foreign protein is correctly folded and oriented in the membrane. Thus, insertion into the inner membrane is a limiting step of the pCoxIV-CYP11A1 topogenesis in yeast cells, whereas its import into mitochondria and proteolytic processing proceed without significant impediments.

[Synthesis in Escherichia coli and certain properties of hybrid protein consisting of a modified precursor of adrenodoxin reductase and shortened precursor of adrenodoxin]. / Sintez v Escherichia coli i nekotorye svoistva gibridnogo belka, sostoiashchego iz modifitsirovannogo predshestvennika adrenodoksinreduktazy i ukorochennogo predshestvennika adrenodoksina.

Some aspects of formation and functioning of the cholesterol hydroxylase system were studied. A hybrid protein was synthesized in E. coli composed of the modified form of the (NADPH)adrenodoxin reductase precursor (N-terminal domain) and the shortened adrenodoxin precursor (C-terminal domain). The modified reductase precursor contained 12 extra amino acid residues at the N-terminus and the N-terminally shortened adrenodoxin precursor had 17 C-terminal amino acids of its targeting presequence. The hybrid reduced cytochrome P450scc in a reconstituted system. Thus, neither the extra 44 amino acids at the N-terminus of the reductase nor the 17 amino acid linker affected the interaction of the active sites in the hybrid protein. These modifications do not interfere with the binding of prosthetic groups and formation of the active sites of two enzymes in the E. coli cells. Modified N-terminal sequence of the hybrid does not affect its import into heterologous mitochondria.

Transgenic yeast expressing human cytochrome P450s can serve as a tool in studies of the mechanisms of their induction by various effectors.

Transgenic Saccharomyces cerevisiae yeast strains were constructed which express CYP2D6 and CYP3A4 genes under control of an artificial promoter. When added to the growth medium, sparteine, a substrate for CYP2D6, was shown to increase the content of this cytochrome P450 isoform in yeast cells. No such increase was observed when a proteinase-deficient yeast mutant was used as a parent strain. Nifedipine, a substrate for CYP3A4, failed to affect the level of CYP3A4 expression even in wild yeast cells. These results suggest that expression of CYP2D6 in human liver can at least partially be controlled post-transcriptionally by its inductors while for CYP3A4 such a mechanism is hardly possible.

Modification of the targeting presequence of the bovine cytochrome P-450scc precursor lifting tissue-specific restrictions on its mitochondrial import.

It has been found that a recombinant cytochrome P-450scc precursor supplemented with an extra MRGSH6GIR sequence at the NH2-terminus (6His-pP450scc) is imported into isolated rat liver and heart mitochondria as well as into yeast mitochondria. The import is coupled with proteolytic processing of the precursor resulting in the mature size form of cytochrome P-450scc. Modification of the targeting presequence responsible for its increased positive charge is supposed to lift the previously suggested tissue-specific restrictions on the pP450scc import into mitochondria.

[Import of a modified form of a cytochrome P450scc precursor into mitochondria from various sources]. / Import modifitsirovannoi formy predshestvennika tsitokhroma P450scc v mitokhondrii iz razlichnykh istochnikov.

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.

High yield expression of functionally active human liver CYP2D6 in yeast cells.

In order to develop a model system for studying drug metabolism, we constructed recombinant yeast strains expressing human liver cytochromes P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimization of the initiation ATG codon context. The PCR-based site-mutagenesis method was used to introduce an AAA sequence immediately before the initiation codon resulting in increased translation of the GAL10-CYC1-derived mRNA. The use of a peptidase-deficient yeast strain also helped to increase the CYP2D6 content. A P450 content of 250 +/- 30 pmol per mg of microsomal protein was achieved. HPLC analysis confirmed that heterologously expressed CYP2D6 catalysed the oxidation of debrisoquine and dextromethorphan, two prototype substrates for CYP2D6. The Km for debrisoquine 4-hydroxylase was found to be 50 microM and Vmax 7.5 pmol mg-1 min-1. Dextromethorphan O-demethylase activity in CYP2D6-containing microsomes was characterized by Km 8.5 microM and Vmax 700 pmol mg-1 min-1. Biotransformation of debrisoquine and dextromethorphan was not detected in control microsomes. Yeast synthesizing CYP2D6 represents a useful in vitro system for studying xenobiotic metabolism.

A modified form of the cytochrome P450scc precursor: a new approach in studies on protein import into mitochondria.

Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells. A large-scale procedure for isolation and purification of this protein was elaborated. 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form. As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria.