The 30-kilodalton subunit of bovine mitochondrial complex I is homologous to a protein coded in chloroplast DNA

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.     

Isolation and structural analysis of ribosomal protein genes in Xenopus laevis. Homology between sequences present in the gene and in several different messenger RNAs

The ribosomal protein genes are present in two to four copies per haploid genome of Xenopus laevis. Using cloned complementary DNA probes, we have isolated, from a genomic library of X. laevis, several clones containing genes for two different ribosomal proteins (L1 and L14). These genes contain intervening sequences. In the case of the L1 gene, the exons are 100 to 200 base-pairs long and the introns, on average, 400 base-pairs. Along the genomic fragments, two different classes of repetitive DNA are present: highly and middle repetitive DNA. Both are evolutionarily unstable as shown by hybridization to Xenopus tropicalis DNA. Several introns of the gene coding for protein L1 contain middle repetitive sequences. Hybridization and hybrid-released translation experiments have shown that sequences inside the two genes hybridize to several poly(A) messenger RNAs. Some of the products encoded by these mRNA have electrophoretic properties of ribosomal proteins.     

A homologue of the nuclear coded 49 kd subunit of bovine mitochondrial NADH-ubiquinone reductase is coded in chloroplast DNA.

The mitochondrial NADH-ubiquinone reductase (complex I) is an assembly of approximately 26 different polypeptides. In vertebrates and invertebrates, seven of its subunits are the products of genes in the mitochondrial DNA, and homologues of these genes have been found previously in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum, although their function in the chloroplast is unknown. The remainder of the subunits of the mitochondrial complex are nuclear gene products that are imported into the organelle, amongst them the 49 kd subunit, a component of the iron--sulphur subcomplex of the enzyme. In the present work, the N-terminal sequence of this protein has been determined, and this has been used to design two mixtures of synthetic oligonucleotides, each containing 32 different sequences 17 bases long. These mixtures have been used as hybridization probes to isolate cDNA clones from a bovine library. The DNA sequences of these clones have been determined and they encode the mature 49 kd protein, with the exception of amino acids 1 and 2. The protein sequence of 430 amino acids is closely related to those of proteins that are encoded in open reading frames (ORFs) present in the chloroplast genomes of M.polymorpha and N.tabacum. Only one cysteine is conserved and the sequences provide no indication that the 49 kd protein contains iron--sulphur centres. These ORFs are found in the single copy regions of chloroplast DNA in close proximity to four of the homologues of the mammalian mitochondrial genes that encode subunits of complex I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.     

Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland

A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland.     

ATP synthase complex from bovine heart mitochondria. Subunit arrangement as revealed by nearest neighbor analysis and susceptibility to trypsin

The nearest neighbor relationships of bovine mitochondrial H(+)-ATPase subunits were investigated by the chemical cross-linking approach using the homobifunctional cleavable reagents dithiobis(succinimidyl propionate) and disuccinimidyl tartrate. Cross-linked proteins were resolved by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Individual subunits were detected by silver staining or by Western blotting and staining with subunit-specific antisera. Products larger than 80,000 daltons were not analyzed. Interactions between F1 subunits included cross-links between gamma and delta as well as gamma and epsilon subunits. Among F0 subunit interactions were observed cross-links of (i) coupling factor 6 (F6) with 8-, 20-, and 24-kDa proteins, (ii) oligomycin sensitivity-conferring protein (OSCP) with 24-kDa protein, and (iii) 20-kDa protein with 24-kDa protein. In addition, several cross-links among subunits involving F1 and F0 sectors were detected. These included cross-links between F6 and alpha, F6 and gamma, OSCP and alpha/beta, and 24-kDa protein and alpha/beta. Thus, OSCP, F6, and the 24-kDa protein were found to form cross-links with both F1 and F0 subunits. The surface accessibility of F0 subunits was investigated by subjecting aliquots of F0 to trypsin treatment. Our data demonstrated that the rate of degradation was in the order OSCP greater than 24-kDa protein greater than or equal to F6 greater than subunit 6. The degradation of subunits of F0 was prevented in intact or reconstituted F1-F0. Based on our present and previously published observations, a model of H(+)-ATPase has been proposed wherein OSCP, F6, and the 24-kDa protein are placed in the stalk region and the alpha and beta subunits of F1-ATPase have been extended down to the membrane surface to enclose the stalk segment.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Mitochondrial NADH:ubiquinone reductase: complementary DNA sequence of the import precursor of the bovine 75-kDa subunit

The 75-kDa subunit of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria is its largest subunit and is a component of the iron-sulfur (IP) fragment of the enzyme. It is encoded in nuclear DNA and is imported into the organelle. Protein sequences have been determined at the N-terminus of the intact protein and on fragments generated by partial cleavage with cyanogen bromide and with Staphylococcus aureus protease V8. Parts of these data have been used to design two mixtures of oligonucleotides 17 bases long, containing 192 and 256 different sequences, which have been synthesized and used as hybridization probes for identification of cognate cDNA clones. Two different but overlapping clones have been isolated, and the sequences of the cloned DNAs have been determined. Together they code for a precursor of the 75-kDa subunit of complex I. The mature protein is 704 amino acids in length, has a calculated molecular mass of 75,961 daltons, and contains no segments of sequence that could be folded into hydrophobic alpha-helixes of sufficient length to span the inner membrane of the mitochondrion. Its precursor has an N-terminal extension of 23 amino acids to specify its import into the mitochondrion from the cytoplasm. Seventeen cysteine residues are dispersed throughout the 75-kDa subunit; some of them are close to each other in the sequence in three separate groups and, by analogy with other iron-sulfur proteins, could be involved in iron-sulfur clusters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Bovine heart mitochondrial F6: HPLC purification, NH2-terminal sequence and the possible structural relatedness to other components of ATPase complexes

The mitochondrial factor F6 has been purified by reverse-phase HPLC and the molecular weight (8500), amino acid composition and about 25% of the amino acid sequence determined. In the NH2-terminal sequence of the first 18 amino acids (NKELDPVQKLFVDKIREY), six identities with the NH2-terminal sequence of the oligomycin-sensitivity conferring protein (OSCP) are apparent, as well as less striking similarities with the OSCP related subunit delta of E. coli F1. The possibility that F6, OSCP and subunit delta of E. coli F1 could have evolved from a common ancestral gene is supported by apparent gene duplication within the OSCP and subunit delta sequences.     

Cloned proteolipid protein and myelin basic protein cDNA. Transcription of the two genes during myelination

cDNA clones of rat brain proteolipid protein (PLP), also named lipophilin, the major integral myelin membrane protein, and of myelin basic protein (MBP), the major extrinsic myelin protein, have been isolated from a rat brain cDNA library cloned into the PstI site of pBR322. Poly(A)+ RNA from actively myelinating 18-day-old rats has been reversely transcribed. Oligonucleotides synthesized according to the established amino-acid sequence of lipophilin and the nucleotide sequence of the small myelin basic protein of the N-terminal, the central and C-terminal region of their sequences were used as hybridization probes for screening. The largest insert in one of several lipophilin clones was 2,585 base pairs (bp) in length (pLp 1). It contained 521 bp of the C-terminal coding sequence and the complete 2,064 bp long non-coding 3' sequence. The myelin basic protein cDNA insert of clones pMBP5 and pMBP6 is 2,530 bp long and that of clones pMBP2 and pMBP3 640 bp. These clones were also characterized. pMBP2 was sequenced and used together with the lipophilin cDNA clones as hybridization probes to estimate the lipophilin and myelin basic protein mRNA levels of rat brain during the myelination period. The expression of the lipophilin and myelin basic protein genes during development of the myelin sheath appears to be strictly coordinated.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Nucleotide sequence of cDNA clones encoding the β subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: The precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension

cDNA and genomic clones encoding the subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79–83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165–168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.     

Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitivity-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase

Early studies have suggested the presence in the central nervous system of possible estrogen binding sites/proteins other than classical nuclear estrogen receptors (nER). We report here the isolation and identification of a 23 kDa membrane protein from digitonin-solubilized rat brain mitochondrial fractions that binds 17beta-estradiol conjugated to bovine serum albumin at C-6 position (17beta-E-6-BSA), a ligand that also specifically binds nER. This protein was partially purified using affinity columns coupled with 17beta-E-6-BSA and was recognized by the iodinated 17beta-E-6-BSA (17beta-E-6-[125I]BSA) in a ligand blotting assay. The binding of 17beta-E-6-BSA to this protein was specific for the 17beta-estradiol portion of the conjugate, not BSA. Using N-terminal sequencing and immunoblotting, this 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP). This protein is a subunit of the FOF1 (F-type) mitochondrial ATP synthase/ATPase required for the coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane to ATP synthesis in the F1 sector of the enzyme. Studies using recombinant bovine OSCP (rbOSCP) in ligand blotting revealed that rbOSCP bound 17beta-E-6-[125I]BSA with the same specificity as the purified 23 kDa protein. Further, in a ligand binding assay, 17beta-E-6-[125I]BSA also bound rbOSCP and it was displaced by both 17beta-E-6-BSA and 17alpha-E-6-BSA as well as partially by 17beta-estradiol and diethylstilbestrol (DES), but not by BSA. This finding opens up the possibility that estradiol, and probably other compounds with similar structures, in addition to their classical genomic mechanism, may interact with ATP synthase/ATPase by binding to OSCP, and thereby modulating cellular energy metabolism. Current experiments are addressing such an issue.     

Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase

Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.