Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2) can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates.
ABSTRACT Members of the mitochondrial carrier family have been reported in eukaryotes only, where they transport metabolites and cofactors across the mitochondrial inner membrane to link the metabolic pathways of the cytosol and the matrix. The genome of the giant virus Mimiviridae mimivirus encodes a member of the mitochondrial carrier family of transport proteins. This viral protein has been expressed in Lactococcus lactis and is shown to transport dATP and dTTP. As the 1.2-Mb double-stranded DNA mimivirus genome is rich in A and T residues, we speculate that the virus is using this protein to target the host mitochondria as a source of deoxynucleotides for its replication.
The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones.
The pathogenesis of the ceroid-lipofuscinoses, inherited storage diseases of children, was studied in an ovine model. This was shown to have clinical and pathological features most in common with the late infantile and juvenile human forms of the disease. The ability to study sequential changes allowed the retinal lesions to be described as a dystrophy of photoreceptor outer segments which preceded loss of the photoreceptor cells. An early decrease in amplitude of the c-wave electroretinograph was attributed to a decrease in the transpigment epithelial component. The decreased a- and b-wave amplitudes were attributed to the changes in and loss of, photoreceptor cells. The chemical components of isolated storage cytosomes were analyzed and shown to consist mostly of protein. Sequence analysis of the dominantly stored protein showed that it was identical to the DCCD reactive proteolipid or subunit c of mitochondrial adenosine triphosphate synthase and that it comprised approximately 50% of storage material. Based on the adage that the dominantly stored species should reflect the underlying biochemical anomaly, it was concluded that it was of pathogenic significance. This highly hydrophobic protein tends to extract with lipids in chloroform/methanol and is thus known as a proteolipid. Some of the remainder of the stored proteins also had this characteristic. It was concluded that ovine ceroid-lipofuscinosis was a proteinosis, more specifically a proteolipid proteinosis and as such it forms the prototype of a new class of storage diseases. Recognition of the nature of the dominantly stored chemical species has helped understanding of a variety of chemical and physical characteristics attributed to the whole pigment.(ABSTRACT TRUNCATED AT 250 WORDS)
Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Iλ represents the extrinsic arm, subcomplex Iα consists of subcomplex Iλ plus part of the membrane arm, and subcomplex Iβ is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Iλ, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex Iβ, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566 ± 2 Da) also detected in the HPLC analysis of subcomplex Iβ. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa. Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Iλ represents the extrinsic arm, subcomplex Iα consists of subcomplex Iλ plus part of the membrane arm, and subcomplex Iβ is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Iλ, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex Iβ, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566 ± 2 Da) also detected in the HPLC analysis of subcomplex Iβ. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa. NADH:ubiquinone oxidoreductase (complex I) (1.Walker J.E. The NADH-ubiquinone oxidoreductase (complex I) of respiratory chains.Q. Rev. Biophys. 1992; 25: 253-324Google Scholar, 2.Weiss H. Friedrich T. Hofhaus G. Preis D. The respiratory-chain NADH dehydrogenase (complex I) of mitochondria.Eur. J. Biochem. 1991; 197: 563-576Google Scholar) catalyzes the first step of the electron transport chain in mitochondria (3.Saraste M. Oxidative phosphorylation at the fin de siècle..Science. 1999; 283: 1488-1493Google Scholar, 4.Schultz B.E. Chan S.I. Structures and proton-pumping strategies of mitochondrial respiratory enzymes.Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 23-65Google Scholar). It transfers electrons from NADH to a non-covalently bound FMN and then via a series of iron-sulfur clusters to the terminal acceptor, ubiquinone. The transfer of two electrons is coupled to the translocation of four protons across the inner membrane (5.Wikström M. Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone.FEBS Lett. 1984; 169: 300-304Google Scholar). The enzyme from bovine heart mitochondria is the best characterized, and it serves as a valuable model for the human enzyme where, because of its involvement in human disease, there is growing interest (6.Smeitink J. Sengers R. Trijbels F. van den Heuvel L. Human NADH:ubiquinone oxidoreductase.J. Bioenerg. Biomembr. 2001; 33: 259-266Google Scholar, 7.Smeitink J. van den Heuvel L. Di Mauro S. The genetics and pathology of oxidative phosphorylation.Nat. Rev. Genet. 2001; 2: 342-352Google Scholar). It is an L-shaped assembly of more than 40 different proteins. Seven hydrophobic components are products of the mitochondrial genome (8.Chomyn A. Mariottini P. Cleeter M.W.J. Ragan C.I. Matsuno-Yagi A. Hatefi Y. Doolittle R.F. Attardi G. Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase.Nature. 1985; 314: 592-597Google Scholar, 9.Chomyn A. Cleeter M.W.J. Ragan C.I. Riley M. Doolittle R.F. Attardi G. URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit.Science. 1986; 234: 614-618Google Scholar), and the remainder are nuclear gene products that are imported into the organelle. One arm of the L-shaped complex is in the plane of the membrane, and the other protrudes into the mitochondrial matrix (10.Grigorieff N. Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I).Curr. Opin. Struct. Biol. 1999; 9: 476-483Google Scholar, 11.Guénebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. Consistent structure between bacterial and mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Mol. Biol. 1998; 276: 105-112Google Scholar). The intact complex has been resolved with chaotropic agents into a number of subcomplexes, and one of them, subcomplex Iλ, represents the extrinsic globular domain of the intact complex (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar, 13.Finel M. Majander A.S. Tyynelä J. Dejong A.M.P. Albracht S.P.J. Wikström M. Isolation and characterisation of subcomplexes of the mitochondrial NADH-ubiquinone oxidoreductase (complex I).Eur. J. Biochem. 1994; 226: 237-242Google Scholar). Subcomplex Iα contains both subcomplex Iλ and part of the membrane arm, and subcomplex Iβ is another independent portion of the membrane arm (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar, 15.Finel M. Skehel J.M. Albracht S.P.J. Fearnley I.M. Walker J.E. Resolution of NADH-ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centres of the enzyme.Biochemistry. 1992; 31: 11425-11434Google Scholar). A long term objective is to determine the atomic structure of bovine complex I, and the definition of the subunit compositions of the intact complex and its subcomplexes is an essential step in this process. In the early 1990s, 35 nuclear encoded subunits were characterized. Since then the purity of the complex has improved, and more sensitive methods for protein analysis have been developed. Therefore, as described below, the subunit compositions of the complex and its subcomplexes have been reanalyzed comprehensively by a combination of fractionation of subunits on 1D 1The abbreviations used are: 1D, one-dimensional; 2D, two-dimensional; ASB-14, amidosulfobetaine-14; IPG, immobilized pH gradient; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 1The abbreviations used are: 1D, one-dimensional; 2D, two-dimensional; ASB-14, amidosulfobetaine-14; IPG, immobilized pH gradient; ESI, electrospray ionization; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. and 2D gels and by HPLC coupled with modern methods of protein analysis by mass spectrometry. The presence in the complex of the 35 previously described subunits has been confirmed, and four hitherto unknown subunits have been detected. The sequences of three of them are described elsewhere (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar, 14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar), and the fourth subunit has not been sequenced yet. It is unlikely that any more subunits of the complex remain to be discovered. The total number of different subunits in the bovine heart complex is 46. The isolation of mitochondria from bovine hearts and the preparation of mitochondrial membranes have been described before (16.Walker J.E. Skehel J.M. Buchanan S.K. Structural analysis of NADH:ubiquinone oxidoreductase from bovine heart mitochondria.Methods Enzymol. 1995; 260: 14-34Google Scholar). Complex I was solubilized with n-dodecyl-β-d-maltoside (Anatrace, Maumee, OH) and purified on a Q-Sepharose HP column (Amersham Biosciences) followed by ammonium sulfate precipitation and gel filtration as before (17.Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Resolution of the membrane domain of bovine complex I into subcomplexes: implications for the structural organization of the enzyme.Biochemistry. 2000; 39: 7229-7235Google Scholar) except that Superose 6 HR was replaced by Sephacryl S-300 HR (Amersham Biosciences). The S-300 column provided an effective way of removing residual cytochrome-c oxidase. All purification steps were carried out at 4 °C. Subcomplexes Iα and Iβ were prepared from complex I by chromatography on Q-Sepharose in 0.1% N,N-lauryldimethylamine oxide with a salt gradient (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). Subcomplexes Iα and Iβ eluted at 260 and 325 mm NaCl, respectively. The breakthrough fractions contained material referred to previously as subcomplex Iγ (see "Results"). The 42-kDa subunit, contaminated with lower levels of other subunits, eluted at 125 mm NaCl. The purification of subcomplex Iλ has been described elsewhere (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar). The subunits of protein complexes were fractionated by SDS-PAGE in 12–22% gels (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar) and on 2D gels (isoelectric focusing followed by SDS-PAGE). For the latter purpose, samples of complex I and its subcomplexes were prepared either by dialysis against a buffer, pH 7.4, containing 20 mm Tris-HCl and 0.05% n-dodecyl-β-d-maltoside followed by concentration to 10–20 mg/ml using Ultrafree-0.5 filter units (Millipore, Bedford, MA) or by precipitation with chloroform/methanol (2:1, v/v) (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). Both methods removed salts from samples, and the chloroform/methanol extraction also removed detergents and lipids. These samples (30–60 μg) were denatured by addition of a solution containing 7 m urea, 2 m thiourea, 1–2% ASB-14 (Calbiochem), dithiothreitol (2.8 mg ml−1), 0.5% IPG buffer (Amersham Biosciences), and a trace of bromphenol blue. Then they were diluted with a similar solution (but without ASB-14) to a final concentration of ASB-14 of 0.15%. Strips of IPG (7 cm, pH 3–10 or 6–11) were rehydrated in these solutions for 12 h at 20 °C with a potential of 20 V. The 2D separations by isoelectric focusing and then SDS-PAGE in a 13% polyacrylamide gel in Tricine buffer (18.Schägger H. von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.Anal. Biochem. 1987; 166: 368-379Google Scholar) were carried out as described previously (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). Subunits of subcomplexes Iα, Iβ, and Iλ were fractionated by reverse phase HPLC on a column of Aquapore RP-300 (PerkinElmer Life Sciences) in 0.1% trifluoroacetic acid with a gradient of acetonitrile (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar). Each peak was collected separately. Every band or spot on 1D or 2D gels was analyzed by peptide mass fingerprinting of tryptic peptides. In every subunit, at least one tryptic peptide was sequenced by tandem MS (see the Supplemental Data Section for more information). The molecular masses of proteins separated by HPLC were measured by ESI-MS (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar, 19.Skehel J.M. Fearnley I.M. Walker J.E. NADH:ubiquinone oxidoreductase from bovine heart mitochondria: sequence of a novel 17.2-kDa subunit.FEBS Lett. 1998; 438: 301-305Google Scholar). Proteins were detected on 1D gels by staining with 0.2% Coomassie R250 in 50% methanol containing 7% acetic acid and on 2D gels with 0.1% colloidal Coomassie G-250 in 3% phosphoric acid and 6% ammonium sulfate. The stained proteins were excised and digested in the gel (20.Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature. 1996; 379: 466-469Google Scholar) at 37 °C with either trypsin (Roche Molecular Biochemicals) in 20 mm Tris-HCl buffer, pH 8.0 containing 5 mm CaCl2 or Asp-N protease (Roche Molecular Biochemicals) in 20 mm Tris-HCl buffer, pH 8.0. Proteins were also digested at room temperature with cyanogen bromide in 70% trifluoroacetic acid (21.van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.J. Mass Spectrom. 2002; 37: 322-330Google Scholar). Cysteine residues were not reduced and alkylated before cleavage. The digests of all subunits were examined in a MALDI-TOF mass spectrometer (TofSpec 2E spectrometer, Micromass, Altrincham, UK) and in many cases also by tandem MS peptide sequencing in a Q-TOF instrument (Micromass) as described previously (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar). The SDS-polyacrylamide gel patterns of complex I and its subcomplexes are shown in Fig. 1. Despite the use of gradient gels, many bands contained more than one subunit especially in the region below 20 kDa. Every band was analyzed by peptide mass fingerprinting, and many were analyzed by tandem MS (see Supplemental Data Part 1, Tables S1-1 and S1-2 and Figs. S1-1 to S1-38). By this means, 42 subunits were identified, and the new subunit, B16.6, was discovered from the SDS-PAGE analysis of subcomplex Iλ (12.Fearnley I.M. Carroll J. Shannon R.J. Runswick M.J. Walker J.E. Hirst J. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).J. Biol. Chem. 2001; 276: 38345-38348Google Scholar). The positions of subunits are shown in Fig. 1. With care, these gel patterns are reproducible, but they are influenced by minor alterations in the composition of the gel. Therefore, they cannot be used as a reliable basis for the precise interpretation of patterns of subunits of bovine complex I separated in other gel systems. Subunits ND1–ND6 and ND4L were the most problematic. They are all very hydrophobic proteins, and so they stain poorly with Coomassie Blue dye, and they tend to form diffuse bands. Subunits ND4L and ND6 were especially difficult. They both migrate in the congested region below 20 kDa, and none of their tryptic peptides was identified. Subunit ND4L was identified from CNBr peptides, and the position of ND6 was determined with a polyclonal antibody (data not shown). The hydrophobic subunit B14.7, which was discovered in the 2D analysis of complex I (see below), also stained weakly with Coomassie Blue dye, and it is possible that its staining is suppressed by co-migration with subunit ND3 (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). However, all of these hydrophobic subunits were detected clearly by silver staining (data not shown). The subunits marked on the gels were found consistently at abundant levels in independent preparations of the various complexes. A number of minor impurities were detected sporadically. They include subunits VB, VIA, and VIB of cytochrome-c oxidase, the Rieske protein of the cytochrome bc1 complex, and two components of the 2-oxoglutarate dehydrogenase complex. In Fig. 1A, it is also apparent that the level of the 42-kDa subunit is substoichiometric. It is lost gradually from the complex during chromatography. Subunit MLRQ was not detected in these analyses. By comparison of Fig. 1, B and C, it is clear that subcomplex Iλ is a fragment of subcomplex Iα. It is also evident that subcomplexes Iα and Iβ represent different and largely non-overlapping subsets of subunits of complex I that together account for most, but not all, of its subunits (Fig. 1, B and D). Other subunits including ND1, ND2, and B14.5b are abundant components of the breakthrough fraction (Fig. 2A). The weakly associated 42-kDa subunit was recovered from the fractionation separately in an almost homogeneous state (Fig. 2B). The subunits of bovine complex I and of its subcomplexes were separated on 2D gels (Fig. 3). By analysis of every spot by tryptic mass mapping and of many by tandem mass spectrometry, 34 of the 45 sequenced subunits of the intact complex were identified, and the new subunit, B14.7, was discovered during the analysis of the intact complex (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). The positions of the subunits on the gels are consistent with calculated isopotential points and molecular masses. The seven hydrophobic subunits ND1–ND6 and ND4L were not detected nor were subunits AGGG, ESSS, and SDAP, all components of the hydrophobic subcomplex Iβ. Their absence illustrates the well known unsuitability of 2D gels for analysis of membrane proteins. Because they are insoluble or at best sparingly soluble in the solutions used for the rehydration of the IPG strips they fail to enter the isoelectric focusing gel. Also subunit MLRQ was not detected in the gels shown in Fig. 3, but in other gels (not shown) this subunit has been identified as a rather indistinct series of spots (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). To a minor extent, the 2D gel patterns were influenced by pretreatment of samples with chloroform/methanol (see "Experimental Procedures"). On the pH 6–11 gel, this pretreatment improved the resolution of subunit B16.6 (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar), diminished the resolution of subunit ASHI, and led to the complete loss of the 10-kDa subunit from subcomplexes. In the gels in Fig. 3, a number of subunits are present as multiply resolved "trains" of spots, which often indicate partial post-translational modifications. Each spot in every train was analyzed by peptide mass fingerprinting, and for each train the MALDI spectra from component spots were very similar. Therefore, the components in each train derive from the same protein, and since the isolated subunits gave unique protein masses by ESI-MS analysis, the trains are artifacts probably arising from partial carbamylation of lysine residues by cyanate derived by disproportionation of urea and/or partial deamidation of asparagines (22.Robinson N.E. Robinson A.B. Molecular clocks.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 944-949Google Scholar, 23.Robinson N.E. Protein deamidation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5283-5288Google Scholar). The subunits of subcomplexes Iα, Iβ, and Iλ, but not of complex I, were resolved by reverse-phase HPLC (see Fig. 4), and the subunits in each peak were identified by ESI-MS (see below) or by SDS-PAGE. The new subunit, ESSS, was discovered in the analysis of subcomplex Iβ (14.Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. Definition of the nuclear encoded protein composition of bovine heart mitochondrial complex I. Identification of two new subunits.J. Biol. Chem. 2002; 277: 50311-50317Google Scholar). In the chromatographic separation of subunits of subcomplexes, the hydrophobic subunits ND1–ND6, ND4L, and B14.7 were not recovered from the column, and therefore their masses were not measured by ESI-MS (see below). In numerous independent analyses of subcomplex Iβ, a protein with a mass of 10,566 (±2) Da has been observed to coelute with subunit SGDH (see Fig. 4C). In subsequent experiments with a modified gradient, the two subunits were resolved partially. The mixture of the two subunits was digested in separate digests with trypsin, endoproteinase Asp-N, and CNBr and with trypsin and CNBr sequentially in a double digest. Only peptides from subunit SGDH were observed. On N-terminal analysis of the mixture, only the sequence of SGDH was observed, but the mass of the unknown subunit is not compatible with it being an N-terminal fragment of SGDH, and therefore its N terminus is modified. The blocking group was not removed by treatment with methanolic HCl. Therefore, the blocking group is not formyl. The absence of an N-formyl group and the molecular mass of the unknown subunit show that the unknown protein is neither an intact ND subunit (mitochondrial DNA gene product) nor a fragment of any of them. For a number of reasons, it cannot be a fragment of other nuclear encoded blocked subunits (B-subunits) of complex I. For example, all of the B-subunits yielded peptides in various digests, and the mass of the unknown subunit is not compatible with it being a fragment of any of them. Therefore, this recalcitrant 46th subunit requires further exploration. The accurate measurement of intact protein masses together with knowledge of the N-terminal sequences of subunits helped to detect and identify many post-translational modifications. All of the proteins were identified by peptide mass fingerprinting of tryptic peptides. Many of the modifications have been described before, and most of them have been verified subsequently by tandem MS experiments. These data will be presented elsewhere. A summary of many of these post-translational modifications is given in Table I and in the following sections.Table IMass measurements by ESI-MS and post-translational modifications of nuclear encoded subunits of bovine complex ISubunitaSubunit names prefixed by B gave no N-terminal sequence by Edman degradation. They have modified ("blocked") N termini. All other subunits gave N-terminal sequences by Edman degradation that have been reported before (1).MassMass differencePost-translational modificationsbΔ import indicates that the DNA sequence encodes an N-terminal extension that acts as a mitochondrial import sequence. This import sequence is not in the mature protein.,cThe 24-kDa subunit is known to contain a [2Fe-2S] cluster (50), and the TYKY subunit contains canonical ligation motifs for two [4Fe-4S] clusters (51). The 51-kDa subunit is thought to contain one [4Fe-4S] cluster (1, 52, 53). The 75-kDa subunit contains 11 conserved cysteines that are likely to ligate one [4Fe-4S] and one [2Fe-2S] cluster (1), although a second [4Fe-4S] cluster has also been suggested (53). The location of the [4Fe-4S] cluster "N2" remains uncertain. It is possible that it is coordinated by three cysteines from the PSST subunit and either a non-cysteine ligand or a fourth cysteine from the 49-kDa subunit (38). In the acidic conditions used for HPLC and electrospray ionization, the Fe-S clusters are lost from the protein, and so they do not influence protein molecular mass measurements.ObservedCalculatedDaDa75 kDaNDdND, not determined.76,960.5NDΔ import, 4Fe-4S, 2Fe-2S51 kDa48,502.548,499.4eCalculated with residue 393 of the 51-kDa subunit and residue 255 of the 42-kDa subunit as tryptophan and lysine.Δ import, 4Fe-4S49 kDa49,198.849,174.6+24.2Δ import,fThe bovine cDNA codes for residue 3 onward, and residues 1 and 2 were determined by direct protein sequencing. The human cDNA sequence encodes a plausible import sequence. Fe-S?30 kDa26,434.226,431.9Δ import24 kDa23,814.823,814.5Δ import, 2Fe-2SPSST20,093.620,077.6+16.0Δ import, 4Fe-4S?TYKY20,194.120,196.0Δ import, 2 × 4Fe-4S42 kDa36,705.036,707.0eCalculated with residue 393 of the 51-kDa subunit and residue 255 of the 42-kDa subunit as tryptophan and lysine.Δ import39 kDa39,122.739,115.1+7.6Δ import18 kDa15,337.515,337.3Δ import15 kDa12,534.412,667.6−133.2−Met13 kDa10,534.410,535.7Δ import10 kDa8,438.38,437.4Δ importAGGG8,493.48,493.4Δ importASHI18,738.318,737.0Δ importESSS14,451.714,453.1Δ importKFYI5,829.05,828.7Δ importMLRQ9,323.39,324.7NoneMNLLgThe cDNA encodes the sequence MMNLL. In the mature protein, methionine 1 is mostly removed. The observed and calculated masses refer to the sequences MMNLL… and MNLL…, respectively. See Supplemental Data Fig. S2-1.6,966.17,097.4−131.3−MetMWFE8,106.08,105.4NonePDSW20,832.720,964.9−132.2−MetPGIV19,959.120,091.2−132.1−MetSDAP10,674.210,109.6+564.6Δ import,hThe bovine cDNA for subunit SDAP was extended in a 5′ direction ∼500 bp beyond the codon for residue 1. This sequence did not contain either a translational initiator or a stop codon in-phase. Also the encoded protein sequence did not have the characteristic features of a mitochondrial import sequence. Therefore, it was concluded that either the 5′ sequence had been added artefactually or that it represented an unspliced intron (M. J. Runswick and J. E. Walker, unpublished results). The corresponding human cDNA appears to encode an import sequence. ACPiACP, acyl carrier protein with serine 44 modified by pantetheine-4′-phosphate with 3-hydroxytetradecanoic acid probably attached via a thioester linkage (see "Results").SGDH16,727.916,726.4Δ importB2221,698.921,788.9−90.0−Met + acetylB1816,477.916,397.8+80.1−Met + myristyljThis subunit contains a canonical myristylation signal nea
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTResolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzymeMoshe Finel, J. Mark Skehel, Simon P. J. Albracht, Ian M. Fearnley, and John E. WalkerCite this: Biochemistry 1992, 31, 46, 11425–11434Publication Date (Print):November 1, 1992Publication History Published online1 May 2002Published inissue 1 November 1992https://pubs.acs.org/doi/10.1021/bi00161a022https://doi.org/10.1021/bi00161a022research-articleACS PublicationsRequest reuse permissionsArticle Views197Altmetric-Citations118LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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