Yeast COQ4 encodes a mitochondrial protein required for coenzyme Q synthesis.

The COQ4 gene coding for a component of the coenzyme Q biosynthetic pathway in the yeast Saccharomyces cerevisiae was cloned by a functional complementation of a Q-deficient mutant strain. Yeast coq4 mutant strains harboring the COQ4 gene on either single- or multicopy plasmids acquired the ability to grow on media containing a nonfermentable carbon source, synthesize Q(6), and respire. COQ4 encodes a polypeptide containing 335 amino acids with a calculated molecular mass of 38.6 kDa. By Western blot analysis with a specific antiserum, Coq4p was shown to peripherally associate with the matrix face of the mitochondrial inner membrane. The putative mitochondrial-targeting sequence present at the amino-terminus of the polypeptide efficiently imported it to mitochondria in a membrane-potential-dependent manner. Steady-state levels of COQ4 mRNA were increased during growth on glycerol-containing medium, in accordance with a function in Q biosynthesis. The function of Coq4p is unknown, although its presence is required to maintain a steady-state level of Coq7p, another component of the Q biosynthetic pathway. The results presented here, along with those available from literature, are discussed in light of the recently proposed existence of a multisubunit complex functioning in Q biosynthesis (A. Y. Hsu, T. Q. Do, P. T. Lee, and C. F. Clarke, 2000, Biochim. Biophys. Acta 1484, 287-297).

The multiple nicotinamide nucleotide-binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P-labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18-20 kDa. The 51 and the 30-kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [beta-32P]ADP. The 42-kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [beta-32P]ADP. It was not labeled with [32P]NADP(H). The 39-kDa polypeptide was labeled with [32P]NADPH and to a small extent with [beta-32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem. 273, 8094-8098]. The 18-20-kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51-kDa subunit is known to contain FMN and a [4Fe-4S] cluster, and is the NAD(P)H-binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide-binding subunits of complex I have been discussed.

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I). Effect of substrates on the fragmentation of subunits by trypsin.

It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >/=20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.

Mitochondrial ATP synthase: Fe2+-catalyzed fragmentation of the soluble F1-ATPase.

The treatment of the soluble F1-ATPase with the Fe2+-ascorbate oxidative system has resulted in the inactivation and fragmentation of the enzyme. Up to 10 polypeptide fragments could be readily observed on the SDS-PAGE. Addition of free Mg2+ or EDTA effectively prevented inactivation and fragmentation. Both alpha and beta subunits of the F1-ATPase were cleaved, with predominant cleavage sites being identified on alpha. Oxidative fragmentation of the F1-ATPase showed nucleotide dependence. Removal of nucleotides from the F1-ATPase as well as their excess in the medium dramatically affected the fragmentation pattern. On the basis of the M(r) of the fragments, their immunorecognition with the antibodies against subunits of the F1-ATPase, and the results of the mild proteolysis of the F1-ATPase with trypsin, cleavage sites are suggested to be located in the nucleotide-binding domain of both alpha and beta subunits. Finally, it is hypothesized that similar structural damage of the F1-ATPase may occur in mitochondrion in vivo under oxidative stress conditions.

Intersubunit interactions in the bovine mitochondrial complex I as revealed by ligand blotting.

Bovine mitochondrial complex I (NADH: ubiquinone oxidoreductase) is composed of 3 structural domains, designated FP (flavoprotein, 3 subunits), IP (iron-sulfur protein, 7-8 subunits) and HP (hydrophobic protein, > 30 subunits). IP intervenes between FP and HP, and in complex I its 75 kDa subunit appears to interact with the 51 kDa subunit of FP. In this study, we show by the technique of ligand blotting that isolated IP binds (a) only to the 51 kDa subunit of FP, and (b) to the 42, 39, 23, 20 and 16 kDa subunits of HP. Because a 23 kDa and a 20 kDa subunit of complex I are potential iron-sulfur proteins, these and our previous results are consistent with the following possible path of electrons in complex I: NADH-->51 and 24 kDa subunit of FP-->75 kDa subunit of IP-->23 and 20 kDa subunits of HP-->ubiquinone.

Membrane topography and near-neighbor relationships of the mitochondrial ATP synthase subunits e, f, and g.

The well characterized subunits of the bovine ATP synthase complex are the alpha, beta, gamma, delta, and epsilon subunits of the catalytic sector, F1; the ATPase inhibitor protein; and subunits a, b, c, and d, OSCP (oligomycin sensitivity-conferring protein), F6, and A6L, which are present in the membrane sector, F0, and the 45-A-long stalk that connects F1 to F0. It has been shown recently that bovine ATP synthase preparations also contain three small polypeptides, designated e, f, and g, with respective molecular masses of 8.2, 10. 2, and 11.3 kDa. To ascertain their involvement as bona fide subunits of the ATP synthase and to investigate their membrane topography and proximity to the above ATP synthase subunits, polyclonal antipeptide antibodies were raised in the rabbit to the COOH-terminal amino acid residues 57-70 of e, 75-86 of f, and 91-102 of g. It was shown that (i) e, f, and g could be immunoprecipitated with anti-OSCP IgG from a fraction of bovine submitochondrial particles enriched in oligomycin-sensitive ATPase; (ii) the NH2 termini of f and g are exposed on the matrix side of the mitochondrial inner membrane and can be curtailed by proteolysis; (iii) the COOH termini of all three polypeptides are exposed on the cytosolic side of the inner membrane; and (iv) f cross-links to A6L and to g, and e cross-links to g and appears to form an e-e dimer. Thus, the bovine ATP synthase complex appears to have 16 unlike subunits, twice as many as its counterpart in Escherichia coli.

ATP synthase complex. Proximities of subunits in bovine submitochondrial particles.

The catalytic sector, F1, and the membrane sector, F0, of the mitochondrial ATP synthase complex are joined together by a 45-A-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F0 and F1. This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits of F1 and F0, as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particles (SMP) and F1-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-linking from zero to 10 A. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits alpha, beta, gamma, delta, OSCP, F6, A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F6 are bound independent of one another. Subunits b, OSCP, d, and F6 cross-linked to alpha and/or beta, but not to gamma or delta. The COOH-terminal half of A6L, which is extramembranous, cross-linked to d but not to any other stalk or F1 subunit. No cross-links of subunits a and c with any stalk or F1 subunits were detected. In F1-deficient SMP, cross-linked b+b and d+F6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. The addition of F1 to F1-deficient particles appeared to reverse these changes. Treatment of F1-deficient particles with trypsin rapidly hydrolyzed away OSCP and F6, fragmented b to membrane-bound 18-, 12-, and 8-9-kDa antigenic fragments, which cross-linked to d and/or with one another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near neighbor relationships of the stalk subunits with one another and with the alpha and beta subunits at a level near the proximal end (bottom) of F1 and at the membrane-matrix interface are presented.

Electron microscopy of two-dimensional crystals of mitochondrial ATP synthase.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and gamma = 86 degrees. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

[Electron microscopic study of ATP synthetase from bovine heart mitochondria]. / Elektronno-mikroskopicheskoe issledovanie ATP-sintetazy iz mitokhondrii serdtsa byka.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at ca. 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a 13.0 nm, b 25.6 nm and gamma 86 degrees. In line with this conclusion, two alternative models for the crystal structural organization are plausible, viz., with one or two protein molecules per unit cell. The first model suggests an asymmetric incorporation of ATP synthase molecules into the lipid bilayer: extramembranous portions F1 are located on one side of the crystal membrane plane. According to the second model, the incorporation occurs on each side of the lipid bilayer, the unit cell containing the two oppositely oriented protein molecules. Based on the absence of another type of the projected crystal images (a rear view of the membrane), unique to the first model, preference is given to the second model.

[Analysis of the trypsin hydrolysate of a 40-kDa protein, found in the nucleoprotein of serum. Identification of it as alpha(1)-acidic glycoprotein (orosomucoid)]. / Analiz produktov tripticheskogo gidrolizata 40-kDa belka, vkhodiashchego v sostav nukleoproteinovoi fraktsii syvorotki krovi. Identifikatsiia ego v kachestve alpha(1)-kislogo glikoproteina (orozomukoida).

From the tryptic hydrolysis of 40-kDa protein from the mouse blood serum thirty-four peptides were isolated by HPLC, of which complete and partial amino acid sequence was established for twenty-eight and six, respectively. On the basis of these data the protein is identified as the blood serum alpha 1-acid glycoprotein.

[Primary structure of the OSCP-protein, conferring the oligomycin sensitivity to the H+-ATPase complex. II. Hydrolysis of OSCP with proteinase from Staphylococcus aureus and reconstruction of the polypeptide chain]. / Pervichnaia struktura OSCP-belka, pridaiushchego mitokhondrial'nomu H+-ATP-aznomu kompleksu chuvstvitel'nost' k oligomitsinu. II. Gidroliz OSCP proteinazoi iz Staphylococcus aureus i rekonstruktsiia polipeptidnoi tsepi belka.

Hydrolysis of OSCP of bovine heart mitochondria by proteinase from Staphylococcus aureus V8 was followed by isolation of all individual peptides by means of gel-filtration and HPLC. Structural analysis of the peptides allowed to arrange BrCN-fragments and to reconstruct the complete amino acid sequence of the protein. Comparative structural analysis revealed existence of a certain homology between OSCP and delta- and b-subunits of the E. coli H+-ATPase, which are necessary for interaction of catalytic and proton-conducting parts of the bacterial enzyme.