Artigo Acesso aberto Revisado por pares

The Post-translational Modifications of the Nuclear Encoded Subunits of Complex I from Bovine Heart Mitochondria

2005; Elsevier BV; Volume: 4; Issue: 5 Linguagem: Inglês

10.1074/mcp.m500014-mcp200

ISSN

1535-9484

Autores

Joe Carroll, Ian M. Fearnley, Mark Skehel, Michael J. Runswick, Richard J. Shannon, Judy Hirst, John E. Walker,

Tópico(s)

Enzyme Structure and Function

Resumo

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-α-acetylated, and a 14th, subunit B18, is N-α-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-α-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I. Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-α-acetylated, and a 14th, subunit B18, is N-α-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-α-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I. Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is one of the most complicated enzymes yet characterized. It is a membrane-bound multisubunit complex with a mass of about 1 MDa (1Walker J.E. The NADH-ubiquinone oxidoreductase (complex I) of respiratory chains.Q. Rev. Biophys. 1992; 25: 253-324Google Scholar). Its structure is L-shaped with one arm lying in the plane of the inner membrane and the other extending into the mitochondrial matrix (2Grigorieff N. Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 Å in ice.J. Mol. Biol. 1998; 277: 1033-1046Google Scholar). Complex I contributes to the conversion of redox energy into the proton motive force by coupling electron transfer from NADH to coenzyme Q to extrusion of protons from the mitochondrial matrix into the intermembrane space. It is thought that transfer of two electrons leads to the translocation of four protons outward across the inner membrane (3Wikström M. Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone.FEBS Lett. 1984; 169: 300-304Google Scholar). The details of these processes are poorly understood, and as part of an effort to unravel the mechanism of complex I, we are characterizing the protein composition of the complex as a prelude to establishing its molecular structure. We have shown that bovine complex I is an assembly of 46 different proteins (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar, 5Fearnley 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, 6Carroll 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, 7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar). Seven hydrophobic proteins are products of the mitochondrial genome (8Anderson S. de Bruijn M.H. Coulson A.R. Eperon I.C. Sanger F. Young I.G. Complete sequence of bovine mitochondrial DNA.J. Mol. Biol. 1982; 156: 683-717Google Scholar, 9Chomyn 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, 10Chomyn A. Cleeter M.W.J. Ragan C.I. Riley M. Doolittle R.F. Attardi G. Last unidentified reading frame of human mtDNA codes for an NADH dehydrogenase subunit.Science. 1986; 234: 614-618Google Scholar). We have characterized the sequences of 38 of the 39 nuclear encoded subunits and determined their distribution between the two arms of the complex (7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar, 11Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. The nuclear encoded subunits of complex I from bovine heart mitochondria.Biochim. Biophys. Acta. 2003; 1604: 135-150Google Scholar). All except two of the 38 sequenced nuclear encoded subunits are modified during or after cytoplasmic translation. Eighteen of the modified subunits have N-terminal extensions acting as mitochondrial import sequences that are removed during the import process. Between four and five of the subunits with processed import sequences also contain iron-sulfur clusters that are involved in electron transfer processes. Other imported subunits do not have processed precursor sequences, but 16 of them lose their translational initiator methionines, and in all except four subunits, the α-amino group of residue 2 becomes modified. Four other subunits retain the initiator methionine. In two of them the α-amino group of this methionine is modified, but in two others it remains unchanged. In addition to being modified N-terminally one subunit (B12) has other modifications in its N-terminal region. The nature of many of the post-translational modifications in bovine complex I has been deduced previously by comparison of accurate molecular mass measurements of intact subunits with their molecular masses calculated from the protein sequences (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar, 12Fearnley I.M. Skehel J.M. Walker J.E. Electrospray mass spectrometric analysis of subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.Biochem. Soc. Trans. 1994; 22: 551-555Google Scholar), but so far, the N-terminal modifying groups of only two subunits (B14.7 and B17.2) have been characterized experimentally (6Carroll 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, 13Skehel 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). In this study, we have established the nature of the blocking groups in the remaining 12 N-terminally modified subunits directly by mass spectrometric analyses of peptides containing the modified residues. We have also confirmed the N-α-acetylation of subunits B14.7 and B17.2. It has been shown that the N-α-amino groups of 13 subunits are acetylated, and a 14th is N-α-myristoylated. In addition, the N-α-acetylated subunit, B12, is a mixture of species with one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations, but the predominant form is methylated on residues 4 and 6. Mitochondria were isolated from bovine hearts (14Smith A.L. Preparation, properties and conditions for assay of mitochondria: slaughterhouse material, small scale.Methods Enzymol. 1967; 10: 81-86Google Scholar), and mitochondrial membranes were prepared as described previously (15Walker 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 purified from mitochondrial membranes by solubilization with n-dodecyl-β-d-maltoside (Anatrace, Maumee, OH) followed by a combination of ion-exchange and gel filtration chromatography (7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar, 16Sazanov 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). The preparation of subcomplexes Iα, Iβ, Iγ, and Iλ has been described elsewhere (5Fearnley 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, 6Carroll 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, 7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar, 16Sazanov 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). The sources of the N-terminally modified subunits of bovine complex I are summarized in Table I. Some subunits were isolated from complex I and its subcomplexes by fractionation on 1D 1The abbreviations used are: 1D, one-dimensional; 2D, two-dimensional. or 2D gels (5Fearnley 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, 7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar). On a 1D gel subunit B14 co-migrated with B14.5a. Other subunits were obtained by reverse phase HPLC on a column of Aquapore RP-300 (2.1 × 100 mm; PerkinElmer Life Sciences) (5Fearnley 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, 7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar) or, in the case of subunit B14.5a, on a PRP-3 column (2.1 × 150 mm; Hamilton Co., Reno, NV) in 0.1% trifluoroacetic acid. Proteins were eluted from the PRP-3 column with a gradient of solvent containing 0.05% trifluoroacetic acid, 40% acetonitrile, and 50% propan-2-ol at a flow rate of 0.1 ml/min. The details of the gradient were as follows: 5–15 min, 0–20% solvent; 15–75 min, 20–80% solvent; 75–85 min, 80–100% solvent. The eluate was monitored at 225 nm. Subunit B13 was obtained by HPLC fractionation of subcomplex Iα on Aquapore RP-300 (7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar) followed by 1D SDS-PAGE of a selected fraction.Table ISources of N-terminally modified subunits of bovine complex ISubunitSourcePurificationB22Iβ1D gelB18Iβ1D gelB17.2Iα1D gelB17Iβ1D gelB16.6Iα1D gelB15Iγ1D gelB14.7Iγ1D gelB14.5aIαHPLCB14.5bIγ1D gelB14Iα1D gelB13IαHPLC and 1D gelB12IβHPLC or 1D gelB9CI2D gelB8IλHPLC Open table in a new tab Proteins in bands and spots excised from gels were digested by "in-gel" cleavage (17Wilm 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 trypsin, chymotrypsin, or endoproteinase Lys-C in buffer consisting of 20 mm Tris-HCl, pH 8.0, and 5 mm calcium chloride, with endoproteinase Asp-N in 20 mm Tris-HCl, pH 8.0, with endoproteinase Arg-C in 24 mm Tris-HCl, pH 8.0, containing 4 mm calcium chloride, 5 mm dithiothreitol, and 0.4 mm EDTA or at room temperature with cyanogen bromide (18van 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). The proteolytic enzymes were purchased from Roche Diagnostics GmbH. Samples of N-terminally modified subunits that had been purified by HPLC were dried and redissolved in 25–100 mm ammonium bicarbonate containing either trypsin (0.3 ng/μl plus 0.4 mm calcium chloride), chymotrypsin (5 ng/μl), or endoproteinase Lys-C (8 ng/μl) and incubated at 37 °C. Digested samples were either stored at −20 °C or acidified with formic acid (0.5–1.0% (v/v) final concentration) before analysis. Peptide digests of subunits of complex I were analyzed in a MALDI-TOF mass spectrometer (TofSpec 2E spectrometer, Micromass, Altrincham, UK) using α-cyano-4-hydroxy-trans-cinnamic acid as the matrix. The instrument was calibrated either with bovine trypsin autolysis products (m/z values, 2163.057 and 2273.16) and a calcium-related matrix ion (m/z value, 1060.048) (5Fearnley 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) or with added peptide standards (renin substrate, angiotensin I, and adrenocorticotropic hormone fragment 18–39 human with protonated molecular masses of 1296.6853, 1758.9331, and 2465.1989 Da, respectively). With the exception of subunit B12, peptide sequences were obtained by tandem MS in an ESI-Q-TOF instrument (Micromass) as described previously (5Fearnley 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). Some digests were introduced by nanoelectrospray; others were prefractionated by liquid chromatography "on-line" to the mass spectrometer (see supplemental data). Samples to be analyzed by nanoelectrospray were desalted first on a ZipTipC18 pipette tip (Millipore) and eluted in 50% acetonitrile containing 1% formic acid. The N-terminal peptides from the tryptic digest of subunit B12 were desalted by hydrophilic interaction chromatography on ZipTipHPL. They were eluted in 50% acetonitrile containing 10 mm formic acid and sequenced by tandem MS following CID with argon in a MALDI-Q-TOF instrument (Micromass) using α-cyano-4-hydroxy-trans-cinnamic acid as the matrix. The instrument was calibrated with the same standards used for calibrating the ESI-Q-TOF instrument (5Fearnley 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). From earlier analyses of intact subunits by Edman sequencing, it was known that 14 of the nuclear encoded subunits of bovine complex I (listed in Table I) have modified N-terminals (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar, 7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar, 13Skehel 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, 19Arizmendi J.M. Skehel J.M. Runswick M.J. Fearnley I.M. Walker J.E. 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?.FEBS Lett. 1992; 313: 80-84Google Scholar). By analyzing various enzyme digests and also cyanogen bromide digests of the subunits by MALDI-TOF mass spectrometry, peptides representing the N-terminal regions of these subunits were identified. From them, N-terminal peptides that were considered to be suitable for mass spectrometric sequencing were selected (see Table II). Then these selected peptides were sequenced by tandem mass spectrometry (see Table II and supplemental data). In the majority of cases (eight of 14), the sequences of the N-terminal peptides were deduced from the C terminus up to residue 2. The masses of residue 1 plus the modifying group and the independent knowledge of the N-terminal residue from the sequences of the subunits allowed the modifying group to be identified (see supplemental data). In one additional case, the mass of the N-terminal dipeptide plus the modifying group and, in another case, the N-terminal tripeptide plus the modifying group allowed the modifying groups to be identified in a similar way. The N-terminal region of subunit B14 was particularly difficult to sequence, and the identification of its modifying group depended on the mass of the N-terminal octapeptide and independent knowledge of the sequence of the subunit in this region. With the exception of subunit B18, which is modified by an N-terminal myristoyl moiety, the other 13 subunits were N-α-acetylated. Molecular mass measurements showed that the N-terminal residues of subunits B14.5b and B12 were modified partially (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar, 12Fearnley I.M. Skehel J.M. Walker J.E. Electrospray mass spectrometric analysis of subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.Biochem. Soc. Trans. 1994; 22: 551-555Google Scholar). This observation was verified for subunit B12 as described below and for subunit B14.5b by analysis of peptides by MALDI-TOF MS (data not shown) and by analysis of the acetylated version by tandem MS (see supplemental data).Table IIMass spectrometric analysis of N-terminally modified peptides from subunits of complex I from bovine heart mitochondriaSubunitPeptideResiduesm/z (z+)Mass (MH+)SequenceObservedCalculatedB22T11–14797.01 (2)1593.021592.81Ac-AFLSSGAYLTHQQKB18T11–7417.83 (2)834.66834.56Myr-GAHLARB17.2T1–21–9594.44 (2)1187.88aThe difference between the observed and calculated values (16 Da) corresponds to the oxidation of the N-terminal methionine residue.1171.69Ac-MELLQVLKRB17R11–9611.18 (2)1221.361221.61Ac-SGYTPEEKLRB16.6M11–10486.40 (2)971.80bThe C-terminal methionine has been converted to homoserine lactone by cyanogen bromide cleavage, leading to the difference (48 Da) between the observed and calculated values. The protonated molecular masses calculated for the modified peptides are 1187.68 and 971.52 Da, respectively.1019.52Ac-AASKVKQDMB15R11–9563.68 (2)1126.361126.55Ac-SFPKYEASRB14.7D11–9603.70 (2)1206.401206.66Ac-AKTVLRQYWB14.5aY1–21–9561.26 (2)1121.521121.57Ac-ASATRFIQWB14.5bY11–11649.56 (2)1298.121297.61Ac-MMTGRQGRATFB14D11–22743.91 (3)2229.732230.23Ac-AASGLRQAAVAASTSVKPIFSRB13T1–21–6336.27 (2)671.54671.45Ac-AGLLKKB9K11–9523.70 (2)1046.401046.60Ac-AERVAAFLKB8K11–12592.05 (2)1183.101182.71Ac-AAAAAIRGVRGKa The difference between the observed and calculated values (16 Da) corresponds to the oxidation of the N-terminal methionine residue.b The C-terminal methionine has been converted to homoserine lactone by cyanogen bromide cleavage, leading to the difference (48 Da) between the observed and calculated values. The protonated molecular masses calculated for the modified peptides are 1187.68 and 971.52 Da, respectively. Open table in a new tab The partial acetylation of subunit B12 was confirmed by the MALDI-TOF spectrum of the tryptic digest of subunit B12 (Fig. 1). The spectrum is dominated by peaks at 1278.608 and 1320.621 corresponding to non-acetylated and acetylated versions of the N-terminal peptide T1, each bearing two methyl groups. On closer inspection, ions corresponding to acetylated and non-acetylated forms of T1 bearing three methyl groups or a single methyl moiety could also be identified at much lower levels than the dimethylated peptides (see Fig. 1, inset). The sequence of peptide T1 contains histidine residues at positions 2, 4, 6, and 8, all of them potential sites for methylation. Therefore, to define the pattern of methylation, the tryptic digest was examined by tandem mass spectrometry in a MALDI-Q-TOF instrument. This analysis allowed the sequences to be determined of each of the various combinations of acetylated and methylated species, permitting the positions of the methylated histidines to be identified. These data are summarized in Table III. The spectra and their interpretations are shown in Fig. 2, and additional details of interpretation are given in the supplementary data.Table IIIMass spectrometric analysis of N-terminal tryptic peptides from subunit B12 of complex I from bovine heart mitochondriaMass (MH+)SequenceObservedCalculated1265.031264.59AHGH*GHEHGPSKaFor both peptides, significant data were observed (see Fig. 2A).AHGHGH*EHGPSKaFor both peptides, significant data were observed (see Fig. 2A).1279.051278.61AHGH*GH*EHGPSK1293.061292.62AHGH*GH*EH*GPSK1292.59Ac-AHGHGHEHGPSKbDeduced from ions of relatively weak intensity in the presence of a stronger second sequence (see Fig. 2, C and D, and the supplemental data section).1307.051306.60Ac-AHGHGH*EHGPSKAc-AHGH*GHEHGPSKbDeduced from ions of relatively weak intensity in the presence of a stronger second sequence (see Fig. 2, C and D, and the supplemental data section).1321.071320.62Ac-AHGH*GH*EHGPSK1335.101334.64Ac-AHGH*GH*EH*GPSKa For both peptides, significant data were observed (see Fig. 2A).b Deduced from ions of relatively weak intensity in the presence of a stronger second sequence (see Fig. 2, C and D, and the supplemental data section). Open table in a new tab Fig. 2.Analysis of modified peptides from the N-terminal region of subunit B12 of bovine complex I. Fragment ions were produced by CID of singly positively charged N-terminal tryptic peptides of subunit B12 in a MALDI Q-TOF mass spectrometer (see Table III). A–F, fragment ion spectra of ions with m/z values of 1265.03, 1279.05, 1293.06, 1307.05, 1321.07, and 1335.10. Ions arising by internal fragmentation are denoted by ♦. Ions arising from isobaric sequences are denoted by □ (see Table III). For explanations of the origins of these fragments and interpretation of the tandem MS data, see the supplemental data section and Supplemental Tables S3–S8.View Large Image Figure ViewerDownload (PPT)Fig. 2.Analysis of modified peptides from the N-terminal region of subunit B12 of bovine complex I. Fragment ions were produced by CID of singly positively charged N-terminal tryptic peptides of subunit B12 in a MALDI Q-TOF mass spectrometer (see Table III). A–F, fragment ion spectra of ions with m/z values of 1265.03, 1279.05, 1293.06, 1307.05, 1321.07, and 1335.10. Ions arising by internal fragmentation are denoted by ♦. Ions arising from isobaric sequences are denoted by □ (see Table III). For explanations of the origins of these fragments and interpretation of the tandem MS data, see the supplemental data section and Supplemental Tables S3–S8.View Large Image Figure ViewerDownload (PPT) These data show that the most abundant methylation pattern is for histidines 4 and 6 to be modified together. There is also evidence for methylation of histidine 4 and histidine 6 separately and of histidines 4, 6, and 8 together, but these species are much less abundant than the form methylated on residues 4 and 6. Both N-α-acetylated and non-acetylated versions of all of these methylation patterns were observed. There was no evidence for other possible patterns of methylation of histidines 4, 6, and 8 or for methylation of histidine 2 alone or in any combination with histidines 4, 6, and 8. Subunit B12 has two additional histidines at C-terminal residues 76 and 77. Neither of them is modified (see Supplemental Table S2). Histidine residues could possibly be methylated on either the N-1 or N-3 nitrogen of the imidazole ring. At present, there is no information about which of these two sites is modified in the methylated histidines in subunit B12. The N-α-acetylation of eukaryotic proteins is one of the most common post-translational modifications, and more than 50%, and possibly as high as 80–90%, of mammalian intracellular proteins are modified in this way (20Tsunasawa S. Sakiyama F. Amino terminal acetylation of proteins: an overview.Methods Enzymol. 1984; 106: 165-170Google Scholar, 21Redman K.L. Rubenstein P.A. Actin amino terminal acetylation and processing in a rabbit reticulocyte lysate.Methods Enzymol. 1984; 106: 179-192Google Scholar). The modification takes place co-translationally after the emergence of 20–30 amino acids of the nascent polypeptide from the ribosome. Often, but not always, it involves the removal of the initiator methionine followed by N-acetylation of residue 2; 78% (11 of 14) of the modified subunits of complex I are acetylated in this manner. Among the complex I subunits investigated here, the initiator methionine is acetylated in subunits B14.5b and B17.2, whereas it has been removed, and residue 2 has been acetylated in all the other subunits that were investigated, excepting subunit B18 (see below). In general, glycine, alanine, serine, methionine, and aspartic acid are dominant as the N-terminal residues of N-α-acetylated proteins (20Tsunasawa S. Sakiyama F. Amino terminal acetylation of proteins: an overview.Methods Enzymol. 1984; 106: 165-170Google Scholar). In bovine complex I, acetylated N-terminal residues are alanine in nine subunits, serine in two subunits, and methionine in two further subunits (see Table II). The 14th modified subunit, B18, is myristoylated on glycine 2 (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar). It has been noted previously that myristoylation occurs only on N-terminal glycine residues after removal of the initiator methionine and that small and uncharged amino acids are preferred at residues 2 and 5 (22Towler D.A. Adams S.P. Eubanks S.R. Towery D.S. Jackson-Machelski E. Glaser L. Gordon J.I. Myristoyl CoA:protein myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities.J. Biol. Chem. 1988; 263: 1784-1790Google Scholar). The N-terminal residue of subunit B18 is glycine, and alanine residues are found at positions 2 and 5, and so the sequence conforms to the canonical myristoylation motif. Why the various subunits of complex I need to be N-terminally acetylated is unclear, but it may be related to the post-translational transfer of the proteins into mitochondria. The biological significance of myristoylation is also obscure. Subunit B18 is a component of subcomplex Iβ, representing part of the membrane arm of complex I, and the myristoyl group may be responsible for binding the subunit to the inner mitochondrial membrane. Methylation of the side chains of basic amino acids is also a common post-translational modification, but methylation of histidine residues is much rarer than methylation of arginine and lysine residues (23Yadav N. Lee J. Kim J. Shen J. Hu M.C.-T. Aldaz C.M. Bedford M.T. Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6464-6468Google Scholar). Among the best known examples are the methylation of histidines to produce 3-methylhistidine in most actins and in myosin heavy chains (24Kalhor H.R. Niewmierzycka A. Faull K.F. Yao X. Grade S. Clarke S. Rubenstein P.A. A highly conserved 3-methylhistidine modification is absent in yeast actin.Arch. Biochem. Biophys. 1999; 370: 105-111Google Scholar, 25Huszar G. Methylated lysines and 3-methylhistidine in myosin: tissue and developmental differences.Methods Enzymol. 1984; 106: 287-291Google Scholar). The actin modification influences its rate of polymerization (26Yao X. Grade S. Wriggers W. Rubenstein P.A. His73, often methylated, is an important structural determinant for actin. A mutagenic analysis of His73 of yeast actin.J. Biol. Chem. 1999; 274: 37443-37449Google Scholar). Otherwise little is known about the biological significance of these histidine modifications in muscle function, although muscle breakdown associated with fasting and athletic activity is monitored by measuring 3-methylhistidine in urine (27Brodsky I.G. Suzara D. Hornberger T.A. Goldspink P. Yarasheski K.E. Smith S. Kukowski J. Esser K. Bedno S. Isoenergetic dietary protein restriction decreases myosin heavy chain IIx fraction and myosin heavy chain production in humans.J. Nutr. 2004; 134: 328-334Google Scholar). The nickel enzyme methyl-coenzyme M reductase, which catalyzes the final step in methane synthesis, contains a 1-methylhistidine residue (28Grabarse W. Mahlert F. Shima S. Thauer R.K. Ermler U. Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation.J. Mol. Biol. 2000; 303: 329-344Google Scholar). The biological significance of the methylation of subunit B12 of complex I is obscure. It is not known at what stage of the synthesis and assembly of the subunit into complex I the modification takes place. This subunit is part of the membrane arm of the complex, and it probably spans the membrane once (4Walker J.E. Arizmendi J.M. Dupuis A. Fearnley I.M. Finel M. Medd S.M. Pilkington S.J. Runswick M.J. Skehel J.M. Sequences of twenty subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria: application of a novel strategy for sequencing proteins using the polymerase chain reaction.J. Mol. Biol. 1992; 226: 1051-1072Google Scholar, 11Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. The nuclear encoded subunits of complex I from bovine heart mitochondria.Biochim. Biophys. Acta. 2003; 1604: 135-150Google Scholar). The locations of the N and C termini have not been established, and so it is not known on which side of the inner mitochondrial membrane the methylation sites lie. In contrast to N-α-acetylation, which appears to be a permanent modification, protein methylation is reversible by demethylases, and methylation-demethylation reactions can have a regulatory function (29Aletta J.M. Cimato T.R. Ettinger M.J. Protein methylation: a signal event in post-translational modification.Trends Biochem. Sci. 1998; 23: 89-91Google Scholar). This aspect of complex I remains to be investigated. The nuclear encoded subunits of bovine complex I contain other significant post-translational modifications that have not been discussed in this study. Around six to eight iron-sulfur clusters are incorporated into four or five other subunits (11Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. The nuclear encoded subunits of complex I from bovine heart mitochondria.Biochim. Biophys. Acta. 2003; 1604: 135-150Google Scholar), and one additional subunit is an acyl carrier protein carrying a phosphopantethenic acid residue on a serine residue (30Runswick M.J. Fearnley I.M. Skehel J.M. Walker J.E. Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria.FEBS Lett. 1991; 286: 121-124Google Scholar). One subunit of the complex with a molecular mass of 10,566 Da has resisted all attempts to sequence it, but its N terminus appears to be modified (7Carroll J. Fearnley I.M. Shannon R.J. Hirst J. Walker J.E. Analysis of the subunit composition of complex I from bovine heart mitochondria.Mol. Cell. Proteomics. 2003; 2: 117-126Google Scholar). Download .pdf (.24 MB) Help with pdf files

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