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M J Runswick

Publications and source records attributed to M J Runswick.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein.

The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.

Carrier Proteins↗
PubMed1996

The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence.

The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.

Adenosine Triphosphate↗
PubMed1996

The F1F0-ATPase complex from bovine heart mitochondria: the molar ratio of the subunits in the stalk region linking the F1 and F0 domains.

The F1 globular catalytic domain and the F0 intrinsic membrane domain of the F1F0-ATPases in bacteria, chloroplasts, and mitochondria are connected by a slender stalk. In the F1F0 complex from bovine heart mitochondria, the stalk is thought to contain subunits OSCP, d, and F6, and the globular part of the membrane bound subunit b, referred to as b'. It has been shown previously that the OSCP, b', d, and F6 proteins can be assembled in vitro into a water soluble complex named the "stalk". The stalk and F1-ATPase together form another complex named F1.stalk. In this paper, the molar ratios of the OSCP, b (or b'), d, and F6 in the stalk, F.stalk, and F1F0-ATPase complexes have been investigated by three independent methods. By quantitation of radioactivity incorporated by S-carboxymethylation with iodo-2-[14C]acetic acid into a stalk complex containing a form of F6 with the mutation Glu3-Cys, it was shown that the stalk consists of equimolar quantities of its four constituent proteins. In the stalk complex containing the natural F6 sequence, this conclusion was confirmed both by quantitation of radioactivity incorporated by Nepsilon-acetimidation with ethyl [1-14C]acetimidate, and by quantitative N-terminal sequence analysis of subunits. By similar Nepsilon-acetimidation experiments, it has been demonstrated that the F1.stalk complex contains one copy per assembly of the OSCP, b', d, and F6 proteins and that the F1F0-ATPase contains one copy per enzyme complex of subunits OSCP, b, and d. The presence of one copy per complex of the OSCP, b' (or b), d, and F6 proteins in the F1.stalk and F1F0-ATPase complexes, respectively, was confirmed by quantitative sequencing.

Acetylation↗
PubMed1996

The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex.

The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits.

Animals↗
PubMed1994

ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein.

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

Adenosine Triphosphatases↗
PubMed1994

ATP synthase from bovine heart mitochondria. In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence.

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli. They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated. In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1. The ratio of b to d in purified F0 was 1:1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

Amino Acid Sequence↗
PubMed1994

Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

Adenosine Triphosphatases↗
PubMed1994

Extension of the mitochondrial transporter super-family: sequences of five members from the nematode worm, Caenorhabditis elegans.

The sequences are presented of cDNAs encoding five related proteins from the nematode worm, Caenorhabditis elegans. Three of them can be recognised as the homologues of the ADP/ATP, phosphate and oxoglutarate/malate carrier proteins that have been found in the inner membranes of mitochondria in other species. These carrier proteins, and the uncoupling protein from the mitochondria in mammalian brown adipose tissue, have common features in their primary and secondary structures, and are members of the same protein super-family. Members of this super-family have polypeptide chains approximately 300 amino acid long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are inter-related, and each repeat is probably folded into a common secondary structural motif consisting of two hydrophobic stretches of amino acids with the potential to form membrane spanning alpha-helices, linked by an extensive hydrophilic region. The common characteristic features of this family of proteins are also present in sequences of two further proteins, named C1 and C2, encoded in nematode cDNAs, and in four published protein sequences from various sources. Neither the transport properties nor the subcellular locations of any of this latter group of six proteins are known. Therefore, currently the super-family of mitochondrial carrier proteins has at least ten different members.

Amino Acid Sequence↗
PubMed1993

F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

Adenosine Triphosphate↗
PubMed1993

The mitochondrial transport protein superfamily.

The ADP/ATP, phosphate, and oxoglutarate/malate carrier proteins found in the inner membranes of mitochondria, and the uncoupling protein from mitochondria in mammalian brown adipose tissue, belong to the same protein superfamily. Established members of this superfamily have polypeptide chains approximately 300 amino acids long that consist of three tandem related sequences of about 100 amino acids. The tandem repeats from the different proteins are interrelated, and probably have similar secondary structures. The common features of this superfamily are also present in nine proteins of unknown functions characterized by DNA sequencing in various species, most notably in Caenorhabditis elegans and Saccharomyces cerevisiae. The high level expression in Escherichia coli of the bovine oxoglutarate/malate carrier, and the reconstitution of active carrier from the expressed protein, offers encouragement that the identity of superfamily members of known sequence but unknown function may be uncovered by a similar route.

Amino Acid Sequence↗
PubMed1992

Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Completion of the primary structure of the complex?

The amino acid sequences of two nuclear-encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N-alpha-amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear-encoded subunits of bovine complex I have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.

Amino Acid Sequence↗
PubMed1992

Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction.

NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequence of the 20 subunits of NADH:ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified alpha-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

NADH: ubiquinone oxidoreductase from bovine heart mitochondria. A fourth nuclear encoded subunit with a homologue encoded in chloroplast genomes.

The amino acid sequence has been determined of the precursor of a nuclear encoded 20 kDa subunit of complex I from bovine heart mitochondria. The sequence of the mature protein is related to a protein of uncertain function, hitherto known as psbG, encoded in the chloroplast genomes of higher plants. Open reading frames encoding homologues of psbG have also been detected in bacteria and in the mitochondrial genome of Paramecium tetraurelia. The chloroplast psbG gene is found between ndhC and ndhJ, which encode homologues of ND3, a hydrophobic subunit of complex I encoded in the bovine mitochondrial genome, and of the nuclear encoded 30 kDa subunit of complex I. This 20 kDa protein is the eleventh out of the forty or more subunits of bovine complex I with a chloroplast encoded homologue, and its sequence provides further support for the presence in chloroplasts of a multisubunit enzyme related to complex I that could be involved in chlororespiration. The strict conservation of three cysteines suggests that the subunit might be an iron-sulphur protein.

Amino Acid Sequence↗
PubMed1992

Sequence and pattern of expression of a bovine homologue of a human mitochondrial transport protein associated with Grave's disease.

A human cDNA has been isolated previously from a thyroid library with the aid of serum from a patient with Grave's disease. It encodes a protein belonging to the mitochondrial metabolite carrier family, referred to as the Grave's disease carrier protein (GDC). Using primers based on this sequence, overlapping cDNAs encoding the bovine homologue of the GDC have been isolated from total bovine heart poly(A)+ cDNA. The bovine protein is 18 amino acids shorter than the published human sequence, but if a frame shift requiring the removal of one nucleotide is introduced into the human cDNA sequence, the human and bovine proteins become identical in their C-terminal regions, and 308 out of 330 amino acids are conserved over their entire sequences. The bovine cDNA has been used to investigate the expression of the GDC in various bovine tissues. In the tissues that were examined, the GDC is most strongly expressed in the thyroid, but substantial amounts of its mRNA were also detected in liver, lung and kidney, and lesser amounts in heart and skeletal muscle.

Amino Acid Sequence↗
PubMed1992

Sequences of the human and bovine genes for the mitochondrial 2-oxoglutarate carrier.

The oxoglutarate carrier transports 2-oxoglutarate across the inner membranes of mitochondria in an electroneutral exchange for malate or other dicarboxylic acids. The sequences of its human and bovine genes have been determined from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the bovine cDNA sequence. The bovine and human genes are split into 6 and 8 exons, respectively, and five introns are found in the same positions in both genes. The coding and protein sequences are 93% and 96.6% identical, respectively. The human oxoglutarate carrier protein is 314 amino acids in length and, in common with the bovine protein, does not appear to have a processed presequence to help to target it into mitochondria.

Amino Acid Sequence↗
PubMed1991

Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria.

The amino-acid sequence of a subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria has been determined and is closely related to those of acyl carrier proteins that are involved in fatty acid biosynthesis in Escherichia coli and plants. Evidence for the presence of covalently attached pantetheine-4'-phosphate in the bovine protein has been obtained by determination of the molecular mass of the isolated subunit by electrospray mass spectrometry, before and after incubation of the protein at alkaline pH under reducing conditions. This decreased the molecular mass from 10,751.6 to 10,449.4, a difference of 302.2 mass units; the value calculated from the protein sequence with one covalently attached pantetheine-4'-phosphate is 10,449.8. The acyl group which is removed by alkaline reduction, appears to be attached via a thioester linkage. By analogy with the bacterial protein it is likely that the attachment site of the pantetheine-4-phosphate is serine-44, which is found in a highly conserved region of the sequence. At present the function of the acyl carrier protein in mitochondrial complex I is not understood.

Acyl Carrier Protein↗