The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.
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
Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.
Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.
Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin.
NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli. NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli. NADH-ubiquinone oxidoreductase (complex I or NDH-1, EC 1.6.5.3) is the first enzyme of the respiratory chains of most mitochondria and many bacteria. It catalyzes the transfer of two electrons from NADH to ubiqinone-10, coupled to the translocation of about 4 protons across the membrane, against the electrochemical concentration gradient (for reviews, see Refs. 1Friedrich T. J. Bioenerg. Biomembr. 2001; 33: 169-177Crossref PubMed Scopus (148) Google Scholar, 2Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar, 3Videira A. Biochim. Biophys. Acta. 1998; 1364: 89-100Crossref PubMed Scopus (82) Google Scholar). Complex I is one of the largest known membrane protein complexes. The bovine enzyme has a mass of ∼980 kDa and is composed of about 46 subunits, including seven hydrophobic ND subunits encoded in the mitochondrial genome (4Carroll J. Shannon R.J. Fearnley I.M. Walker J.E. Hirst J. J. Biol. Chem. 2002; 277: 50311-50317Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The simplest version of the complex in terms of protein content is the prokaryotic enzyme, which has 13-14 subunits and a combined molecular mass of about 550 kDa (5Yagi T. Yano T. DiBernardo S. Matsuno Yagi A. Biochim. Biophys. Acta. 1998; 1364: 125-133Crossref PubMed Scopus (204) Google Scholar). Complex I is currently the least understood component of the respiratory chain. In contrast to other enzymes of the respiratory chain, the atomic structure of complex I is not known and as a consequence, the mechanisms of proton pumping and electron transfer are also not established. Electron microscopy has shown that the mitochondrial as well as the bacterial enzyme have a characteristic L-shaped structure. One arm is embedded in the membrane and the other, the peripheral arm, protrudes into the mitochondrial matrix or bacterial cytoplasm. The first structural model of complex I derived from two-dimensional crystals of the Neurospora crassa enzyme in negative stain was at about 30-Å resolution (6Leonard K. Haiker H. Weiss H. J. Mol. Biol. 1987; 194: 277-286Crossref PubMed Scopus (86) Google Scholar, 7Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar). The highest resolution three-dimensional model is currently the 22-Å structure of bovine complex I produced by single particle averaging of molecules embedded in vitrified ice (8Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (299) Google Scholar). Complex I from Escherichia coli is similar in size and shape to mitochondrial enzymes, although it is thinner, as indicated by single particle analysis of negatively stained samples (9Guenebaut V. Schlitt A. Weiss H. Leonard K. Friedrich T. J. Mol. Biol. 1998; 276: 105-112Crossref PubMed Scopus (204) Google Scholar, 10Guenebaut V. Vincentelli R. Mills D. Weiss H. Leonard K.R. J. Mol. Biol. 1997; 265: 409-418Crossref PubMed Scopus (131) Google Scholar). This established view has been challenged recently by a proposal that active complex I adopts a different, "horseshoe"-like conformation and that the accepted L-shape is an artifact because of solubilization by detergent. This alternative conformation would arise from folding of the peripheral arm toward the distant end of the membrane arm (11Bottcher B. Scheide D. Hesterberg M. Nagel-Steger L. Friedrich T. J. Biol. Chem. 2002; 277: 17970-17977Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). All of the subunits of a minimal complex I proposed from known sequences (2Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar) have analogues in the bacterial enzyme, which is considered to be the simplest version of the enzyme. Dissociation of complex I by chaotropes and detergents indicated that all the redox centers of the enzyme (flavin mononucleotide and up to 8-9 iron-sulfur clusters) are in the peripheral hydrophilic arm (2Walker J.E. Q. Rev. Biophys. 1992; 25: 253-324Crossref PubMed Scopus (681) Google Scholar, 12Friedrich T. Biochim. Biophys. Acta. 1998; 1364: 134-146Crossref PubMed Scopus (179) Google Scholar, 13Finel M. Skehel J.M. Albracht S.P. Fearnley I.M. Walker J.E. Biochemistry. 1992; 31: 11425-11434Crossref PubMed Scopus (130) Google Scholar, 14Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar). The membrane arm is composed of highly hydrophobic subunits and some of these are likely to participate in proton pumping. Sequence comparisons suggested that the hydrophobic subunits ND2, ND4, and ND5 (bovine nomenclature) evolved from a common ancestor related to subunits of K+ or Na+/H+ antiporters (1Friedrich T. J. Bioenerg. Biomembr. 2001; 33: 169-177Crossref PubMed Scopus (148) Google Scholar, 15Fearnley I.M. Walker J.E. Biochim. Biophys. Acta. 1992; 1140: 105-134Crossref PubMed Scopus (291) Google Scholar, 16Kikuno R. Miyata T. FEBS Lett. 1985; 189: 85-88Crossref PubMed Scopus (38) Google Scholar). Further disruptions indicated that subunits ND4 and ND5 are likely to be situated in the part of the membrane arm distal from the peripheral arm (14Sazanov L.A. Peak-Chew S.Y. Fearnley I.M. Walker J.E. Biochemistry. 2000; 39: 7229-7235Crossref PubMed Scopus (157) Google Scholar). This was confirmed by our electron crystallography studies, which located subunit ND5 to the distal end of the membrane arm (17Sazanov L.A. Walker J.E. J. Mol. Biol. 2000; 302: 455-464Crossref PubMed Scopus (63) Google Scholar). These findings led us to suggest that the proton-pumping machinery of complex I might involve a combination of two mechanisms: direct (redox-driven) and indirect (conformation-driven). A more detailed discussion of such a two-mode combination is given in a recent review (1Friedrich T. J. Bioenerg. Biomembr. 2001; 33: 169-177Crossref PubMed Scopus (148) Google Scholar). A high-resolution structure of the enzyme is necessary to establish a detailed mechanism of complex I function, and this is particularly important in view of the increasing number of human disorders associated with mutations in complex I subunits (18Schapira A.H. Biochim. Biophys. Acta. 1998; 1364: 261-270Crossref PubMed Scopus (151) Google Scholar). As can be expected for a large membrane protein, three-dimensional crystals of complex I are extremely difficult to obtain, whereas two-dimensional crystallization is more feasible. Previously, the only reported two-dimensional crystals of intact complex I were obtained with the N. crassa enzyme and studied in negative stain, which limits the resolution to about 20-25 Å (6Leonard K. Haiker H. Weiss H. J. Mol. Biol. 1987; 194: 277-286Crossref PubMed Scopus (86) Google Scholar, 7Hofhaus G. Weiss H. Leonard K. J. Mol. Biol. 1991; 221: 1027-1043Crossref PubMed Scopus (166) Google Scholar). We have produced two-dimensional crystals of major subcomplexes of bovine complex I and reported a projection map of frozen-hydrated crystals at 13-Å resolution (17Sazanov L.A. Walker J.E. J. Mol. Biol. 2000; 302: 455-464Crossref PubMed Scopus (63) Google Scholar). However, it has proved difficult to obtain two-dimensional crystals of the intact bovine complex. Additionally, the medium-resolution projection maps of the mitochondrial enzyme are difficult to interpret because of the large number of subunits (more than 20 subunits in the membrane arm alone). As the bacterial enzyme is much simpler than the mitochondrial one, but is expected to have a similar mechanism, we decided to utilize it for structural studies, using E. coli as a model system. This report contains the structural and biochemical characterization of E. coli complex I purified by an improved procedure from BL21 cells. The activity of the enzyme toward decyl-ubiquinone was fully restored by the addition of native E. coli lipids. Two different forms of two-dimensional crystals were obtained using lipids containing native E. coli extracts. The crystals were studied by electron microscopy in negative stain and found to contain intact, L-shaped complex I. Single particle analysis and activity assays have shown that active complex I maintains an L-shape in detergent solution. These results suggest that complex I is L-shaped in vivo, and indicate a role for native lipids in the activation, stabilization, and crystallization of purified complex I from E. coli. Chemicals—Dodecylmaltoside (DDM) 1The abbreviations used are: DDM, dodecylmaltoside; CMC, critical micelle concentration; DOPC, dioleoylphosphatidylcholine; EPL, E. coli polar lipids; ETL, E. coli total lipid extract; FeCy, ferricyanide; dNADH, deamino-NADH; MES, 2-(N-morpholino)-ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. was purchased from Anatrace (Maumee, OH), taurodeoxycholate and dioleoylphosphatidylcholine (DOPC) from Fluka (Gillingham, Dorset, United Kingdom), other detergents from Calbiochem (Nottingham, UK), Complete protease inhibitors tablets were obtained from Roche Diagnostics (Lewes, UK), Bio-scale DEAE column and Bio-Beads from Bio-Rad (Hemel Hempstead, Herts., UK), other chromatography columns from Amersham Biosciences, and E. coli total and polar lipid extract from Avanti Polar Lipids (Alabaster, AL). All other chemicals were purchased from Sigma. Bacterial Growth and Membrane Preparation—E. coli strain BL21 was grown from a 0.3-liter overnight culture in a 30-liter fermentor at 37 °C and 2% dissolved oxygen in Luria-Bertani (LB) media for about 24 h until late exponential stage (A600 ∼ 3.0). Cells (∼ 180 g) were harvested by centrifugation at 3900 × g (average value here and throughout) for 20 min, re-suspended in RB buffer (50 mm MES, pH 6.0) and collected by centrifugation at 6800 × g for 15 min. When necessary, cells were kept frozen as a pellet at -20 °C. For a small scale preparation, the cells were grown in three-fourths full 2-liter flasks with slow (140 rpm) agitation. Cells (120 g wet weight) were re-suspended in ∼500 ml of RBP buffer (RB + 0.002% phenylmethylsulfonyl fluoride, always added fresh from a 1% stock in absolute ethanol). The suspension was passed twice through a Z-plus 2.2 kilowatt cell disruptor (Constant Systems Ltd.) at 30,000 p.s.i. Cell debris was removed by centrifugation at 9,600 × g for 15 min followed by 18,800 × g for 25 min. The supernatant was centrifuged for 4 h at 150,000 × g. The brown membrane pellets were re-suspended in an ∼150 ml total volume of RBP buffer (RB buffer with Complete EDTA-free protease inhibitors tablets added according to the manufacturer's instructions) using a glass-Teflon homogenizer. When necessary, membranes were stored at -80 °C. Purification of Complex I—Membranes from the equivalent of 120 g of cells were used for a single purification. DDM from 10% stock in water was added dropwise (under constant stirring and on ice) to the membrane suspension to a final concentration of 2%. After a 1-h incubation, non-solubilized material was removed by centrifugation for 1 h at 150,000 × g. The supernatant was passed through a 0.45-micron filter and adjusted to 150 mm NaCl by adding dropwise a solution of 1 m NaCl. At this stage, all deamino-NADH:ferricyanide (FeCy) activity present in the membranes was found in solution (about 8000 μmol of dNADH oxidized per min). The ÄKTA Explorer chromatography system (Amersham Biosciences) was used, with monitoring at 280/420/605 nm to follow absorbance changes because of co-factors present in various proteins. A HiLoad 26/10 Q-Sepharose column was equilibrated with buffer A (20 mm MES, pH 6.0, 0.1% DDM, 10% glycerol, 0.002% phenylmethylsulfonyl fluoride). Solubilized membranes were applied and eluted at 1 ml/min using 60 ml of 15-20% linear gradient of buffer B (A with 1 m NaCl) in buffer A, followed by 800 ml of 20-35% linear gradient of buffer B in buffer A. Complex I eluted at about 500 ml of the elution volume (∼280 mm NaCl), as judged from the dNADH:FeCy activity of fractions. Active fractions (about 150 ml) were pooled, diluted with an equal volume of buffer A, and applied to a Bio-Scale DEAE 20 column equilibrated with buffer A. Retained material was eluted with 10 ml of 0-15% linear gradient of buffer B in buffer A, followed by 600 ml of 15-30% linear gradient of buffer B in buffer A. Complex I eluted as a first peak at about 280 ml (as judged from the dNADH:FeCy activity and SDS-PAGE profiles of the fractions), well separated from the second peak at about 450 ml, which is likely to be one of the terminal oxidases. Fractions containing complex I were pooled (about 180 ml) and concentrated on Vivacell 70 (100 kDa cut-off) concentrators to a volume of about 2 ml. A HiLoad Superdex 200 16/60 Prep Grade gel filtration column was equilibrated with 20 mm MES, pH 6.0, 200 mm NaCl, 2 mm CaCl2, 0.5% DDM, 10% glycerol, 0.002% phenylmethylsulfonyl fluoride. The concentrated sample was applied and eluted at a flow rate of 0.5 ml/min. Fractions of 1.5 ml were collected (at this stage, all dNADH:FeCy activity co-eluted with the main protein peak), analyzed by SDS-PAGE, and those containing pure complex I were pooled and diluted with buffer A to reduce the NaCl concentration to about 50 mm. Then the sample was concentrated to about 6-8 mg/ml using Vivaspin 20 (100 kDa cut-off) concentrators. Purified complex I was stored in small aliquots under liquid nitrogen. Identification of the Subunits of the Complex—Proteins resolved by SDS-PAGE and stained with 0.1% colloidal Coomassie G-250 (3% phosphoric acid, 6% ammonium sulfate) were identified by peptide mass fingerprinting and tandem MS peptide sequence data. Excised gel bands were digested "in-gel" (19Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar) with trypsin (20 mm Tris-HCl, pH 8, 5 mm CaCl2, 37 °C) or CNBr (20van Montfort B.A. Canas B. Duurkens R. Godovac-Zimmermann J. Robillard G.T. J. Mass Spectrom. 2002; 37: 322-330Crossref PubMed Scopus (80) Google Scholar), without prior reduction and alkylation. Portions of the digest were examined in positive ion mode by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with a TofSpec 2E instrument (Micromass, Altrincham, UK) in the presence of α-cyano-4-hydroxycinnamic acid as matrix. Spectra were calibrated with trypsin autolysis peptides (2163.057 and 2273.160) and a matrix-related ion (1060.048). Peptide mass data were screened against databases of protein sequences using the ProteinProbe program (Micromass). Peptide sequence analysis was performed on a Q-TOF mass spectrometer equipped with ESI (Micromass) and coupled on-line to a capillary high performance liquid chromatography (CapLC; Micromass). Peptide mixtures were separated using a PepMap C18 column (180 μm × 100 mm, LC Packings, Amsterdam, The Netherlands) with an acetonitrile gradient in 0.1% formic acid. Acquired tandem MS spectra were interpreted manually, assembled into Peptide Sequence Tags (21Mann M. Wilm M. Anal. Chem. 1994; 66: 4390-4399Crossref PubMed Scopus (1317) Google Scholar), and compared with protein sequence databases. Two-dimensional Crystallization—Lipids in organic solvents were mixed to the desired proportion, washed in chloroform and diethyl ether, dried under a stream of nitrogen, and resuspended in a buffer containing 20 mm MES, pH 6.0, with 2% detergent. For crystallization, the enzyme (0.5 mg/ml final concentration) was mixed with lipid (lipid/protein ratio, 0.3-0.4) in an Eppendorf tube, with the addition of other stock solutions to achieve the desired conditions (0.1 m MES, pH 6.5, 20-40 mm NaCl, 5-10 mm CaCl2). Bio-Beads SM-2 (15 mg/mg of total amount of detergent present in a set-up) were added to allow for slow (about 6-10 h) removal of detergent (22Rigaud J.L. Mosser G. Lacapere J.J. Olofsson A. Levy D. Ranck J.L. J. Struct. Biol. 1997; 118: 226-235Crossref PubMed Scopus (176) Google Scholar). The samples were flushed with nitrogen, sealed, and kept at the desired temperature. Electron Microscopy and Image Processing—For single particle analysis, protein was diluted to 10 μg/ml in buffer as indicated in the figure legends and applied to carbon-coated copper grids (glow-discharged in air). After a 2-min incubation, excess buffer was removed by blotting, the grid was washed twice with the same buffer containing no protein or detergent and then stained with either 2% uranyl acetate or 1% gold thioglucose for 10 s. Crystals were stained with 2% uranyl acetate. Images were recorded with a Philips Tecnai 12 microscope operating at 120 kV and magnification of ×42,000, on Kodak SO163 film. Electron micrographs were checked for astigmatism using an optical diffractometer. Good crystalline areas (or whole films for single particle analysis) were digitized on a Zeiss-SCAI scanner at a 7-μm step size (corresponding to 1.67 Å at the specimen level) and demagnified by linear interpolation on the computer to obtain a pixel size corresponding to 3.33 Å at the specimen level for crystals and to 5 Å for single particles. Individual images of crystals were corrected for long-range disorder and projection maps calculated using MRC (23Henderson R. Baldwin J.M. Ceska T.A. Zemlin F. Beckmann E. Downing K.H. J. Mol. Biol. 1990; 213: 899-929Crossref PubMed Scopus (2526) Google Scholar, 24Crowther R.A. Henderson R. Smith J.M. J. Struct. Biol. 1996; 116: 9-16Crossref PubMed Scopus (665) Google Scholar) and CCP4 (25CCP4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) software suites. The defocus of images was estimated using program CTFFIND2 (8Grigorieff N. J. Mol. Biol. 1998; 277: 1033-1046Crossref PubMed Scopus (299) Google Scholar). Some of the MRC programs were modified to accommodate large unit cells. Single particle analysis was performed with the IMAGIC 5 software package (26van Heel M. Harauz G. Orlova E.V. Schmidt R. Schatz M. J. Struct. Biol. 1996; 116: 17-24Crossref PubMed Scopus (1049) Google Scholar). In total, 500 particles were picked (without selection for appearance) and boxed off from the micrographs of complex I that had been incubated for 2 days at 4 °C with 50 mm NaCl in the buffer and then stained with uranyl acetate. The particle images were normalized and band-pass filtered using a low frequency cut-off of 1/500 Å-1 and a high frequency cut-off of 1/15 Å-1 and centered by translational alignment to a rotational averaged total sum. The centered particle images were grouped into 24 classes using multivariate statistical analysis. All 24 class-sum images were used as references for a multireference alignment and the process was iterated until stable class-sum images were obtained. After the final iteration, aligned particle images were grouped into 6, 12, or 24 classes using multivariate statistical analysis and class-sum images were calculated and compared. Analytical Methods—Protein concentrations were determined by the Bradford (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar) (Bio-Rad) method with bovine serum albumin as standard. Pre-prepared Tris glycine-polyacrylamide gels containing a 10-20% acrylamide gradient were used according to the manufacturer's instructions (Novex). Enzyme activity assays were performed at 30 °C in a Shimadzu UV-1601 spectrophotometer with a magnetic stirrer attachment. Reduction of FeCy was followed at 420 nm, and oxidation of NADH in the presence of decyl-ubiquinone, at 340 nm. All reactions in the presence of lipids or quinones were observed under constant stirring. Reactions were started by addition of 1-2 μl of protein to a 2-ml assay mixture, except for assays in the presence of lipids or quinones, when all the ingredients, except for NADH, were incubated for 2 min at 30 °C with stirring, and the reaction was started by addition of NADH. The rates were estimated using the steady-state (linear) part of the time course of the reaction. Analytical gel-filtration chromatography was performed using a Superose 6 column equilibrated with a buffer containing 20 mm MES, pH 6.0, 200 mm NaCl, 2 mm CaCl2, 0.1% DDM, 10% glycerol, 0.002% phenylmethylsulfonyl fluoride at a flow rate of 0.3 ml/min. Fractions of 0.5 ml were collected and analyzed by SDS-PAGE. The column was calibrated using a high molecular weight gel filtration calibration kit (Amersham Biosciences). Purification and Subunit Composition of Complex I—An increase in the levels of enzyme in the E. coli membranes is required to obtain sufficient amounts of purified complex I for structural studies. We opted for the induction of its expression by limited oxygen availability as an alternative to overexpression by promoter replacement (27Spehr V. Schlitt A. Scheide D. Guenebaut V. Friedrich T. Biochemistry. 1999; 38: 16261-16267Crossref PubMed Scopus (42) Google Scholar). It is known that under aerobic conditions complex I expression decreases in exponential and stationary phases of E. coli growth (28Wackwitz B. Bongaerts J. Goodman S.D. Unden G. Mol. Gen. Genet. 1999; 262: 876-883Crossref PubMed Scopus (45) Google Scholar), when the non-coupling, non-proton pumping dehydrogenase NDH-2 is expressed and used preferentially (29Unden G. Bongaerts J. Biochim. Biophys. Acta. 1997; 1320: 217-234Crossref PubMed Scopus (536) Google Scholar). An optimal level of complex I induction was achieved in Luria-Bertani medium at 2% dissolved oxygen. Under these conditions the activity of complex I in the cytoplasmic membranes (assayed with a specific complex I substrate deamino-NADH and FeCy) was about 1.5-2 μmol/min/mg of protein, 2-3 times higher than during any stage of aerobic growth. Deamino-NADH:FeCy activity was about 80% of NADH:FeCy activity, indicating that only low amounts of NDH-2 were present in our preparations. The purification procedure (Fig. 1) was developed using elements of previously described methods (27Spehr V. Schlitt A. Scheide D. Guenebaut V. Friedrich T. Biochemistry. 1999; 38: 16261-16267Crossref PubMed Scopus (42) Google Scholar, 30Leif H. Sled V.D. Ohnishi T. Weiss H. Friedrich T. Eur. J. Biochem. 1995; 230: 538-548Crossref PubMed Scopus (254) Google Scholar), but with different chromatography steps and avoiding NaBr treatment of the membranes and associated loss of protein. The final gelfiltration step produced a pure preparation with a molecular weight of about 600,000 (by comparison with molecular weight standards), indicating that the complex is monodisperse (Fig. 1C). Notably, a contaminant of about 150 kDa that was present throughout the purification could be removed only when the final gel-filtration step was conducted with 0.2 m NaCl in the buffer. This protein was identified as subunit NuoCD by peptide mass mapping and is likely to be a multimer of this hydrophilic subunit. The significance of this finding remains to be established; it is possible that NuoCD is transiently present as a multimer during assembly of complex I. Previously, co-expression of NuoCD was found to be essential for overexpression and assembly of an NADH dehydrogenase fragment containing NuoE, -F, and -G in E. coli (31Braun M. Bungert S. Friedrich T. Biochemistry. 1998; 37: 1861-1867Crossref PubMed Scopus (69) Google Scholar). A summary of the purification procedure is given in Table I.Table IPurification of complex I from E. coli BL21 Starting material was 120 g (wet weight) of cells. FeCy reduction was followed with dNADH as electron donor.Purification stepVolProteinTotal activitySpecific activitymlmgμmol min-1μmol min-1 mg-1Membrane fraction100348076002.2Solubilized membranes110154074004.8Q-Sepharose150190490025.8DEAE17062280045.2Superdex 200938220057.9 Open table in a new tab All 13 subunits encoded by the nuo operon have been positively identified in the preparation by mass spectrometry using a combination of peptide mass mapping and tandem MS analyses (Fig. 1D, Table II). No significant contaminants were identified on Coomassie-stained gels (Fig. 1D). For some of the highly hydrophobic subunits (NuoL, -M, and -J) few tryptic peptides were recovered and reliable identifications were possible only after tandem MS analyses of the peptides. Table II contains data on sequences of tryptic peptides recovered for the hydrophobic subunits. These peptides are likely to originate from hydrophilic loops and might be useful in secondary structure predictions. Two subunits, NuoE and NuoJ, are not resolved by our gel system, whereas NuoM and NuoN run close to each other, but are partially separated. The relative positions of some subunits differ from those previously reported (27Spehr V. Schlitt A. Scheide D. Guenebaut V. Friedrich T. Biochemistry. 1999; 38: 16261-16267Crossref PubMed Scopus (42) Google Scholar, 32David P. Baumann M. Wikstrom M. Finel M. Biochim. Biophys. Acta. 2002; 1553: 268-278Crossref PubMed Scopus (16) Google Scholar), probably because of slight differences in the gel systems used. We have not observed two separate bands for the NuoI subunit as reported recently (32David P. Baumann M. Wikstrom M. Finel M. Biochim. Biophys. Acta. 2002; 1553: 268-278Crossref PubMed Scopus (16) Google Scholar), although the NuoI band is broad and partially overlaps the NuoE/J band (Fig. 1D).Table IIIdentification by mass-spectrometry of subunits present in E. coli complex I Data are for tryptic peptides.Subunit (SWISS-PROT accession No.)Peptide mass fingerprint dataTandem MS dataNo. of peptide masses matchedSequence coverageSequenceaIdentified sequences within peptides are underlinedMH+1 (monoisotopic)MH+1 (sequence)NuoG (P33602)2539.0%NuoCD (P78089)2549.8%NuoF (P31979)1538.0%GGAGFSTGLK894.48894.47NLEEFFAR1025.501025.51GEYIEAAVNLR1234.601234.64TPETHPLTWR1237.631237.63AIAEATEAGLLGK1243.661243.69EILEDYAGGMR1253.541253.58ALTGLSPDEIVNOVK1583.781583.86NuoL (P33608)No significant dataGLLLSENGYLR1234.661234.68TLVTSIANSAPGR1286.681286.71NuoM (P31978)No significant dataTAAYGLLR864.50864.49LGFFIAFAVKMbProducts of CNBr cleavage, the mass difference observed corresponding to conversion of C-terminal Met to homoserine-lactone1195.661243.69NuoN (P33608)38.2%GPDADSLFSYR1227.561227.56(188-196, 293-303, 368-382)cResidue numbers of tryptic peptides observed by peptide mass fingerprinting of hydrophobic subunitsALVYAQSGDLSFVALGKNLGDGMbProducts of CNBr cleavage, the mass difference observed corresponding to conversion of C-terminal Met to homoserine-lactone2278.202326.18NuoH (P33603)716.0%LLGLFQNR960.58960.56(38-46, 39-46, 52-66, 67-76dBoth unmodified and Met-oxidized peptides were observed 141-148, 294-303)cResidue numbers of tryptic peptides observed by peptide mass fingerprinting of hydrophobic subunitsNuoB (P33598)834.6%MAPVJOR814.46814.46MDYTLTR899.44899.43QEIVTDPLEQEVNK1641.801641.83IDPNGENDRYPLQK1658.801658.81NuoI (P33604)735.0%YPEYNFYR1151.481151.52ELLVGFGTQVR1218.601218.68MYPEEPVYLPPR1490.621490.74NuoE (P33601)628.3%AASIEALK802.46802.47LNIKPGQTTFDGR1446.761446.77MHENQQPQTEAFELSAAER2215.932216.00NuoJ (P33605)No significant dataAGEVLSNR845.44845.45NuoA (P33597)627.2%IGALDWTPAR1099.581099.59(45-61, 47-61, 120-129, 135-145, 133-145dBoth unmodified and Met-oxidized peptides were observed)cResidue numbers of tryptic peptides observed by peptide mass fingerprinting of hydrophobic subunitsMNPETNSIANR1246.561246.59NVPFESGIDSVGSAR1534.661534
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