Definition of the mitochondrial proteome by measurement of molecular masses of membrane

Joe Carroll, Ian M. Fearnley, and John E. Walker*

Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, United Kingdom

Contributed by John E. Walker, September 4, 2006 The covalent structure of a is incompletely defined by its by gel filtration in organic solvents before measurement of intact sequence, and mass spectrometric analysis of the intact protein masses (6–8). protein is needed to detect the presence of any posttranslational We have devised a different approach that avoids the use of modifications. Because most membrane proteins are purified in detergents altogether, where the proteins are extracted from detergents that are incompatible with mass spectrometric ioniza- membranes in organic solvents and then fractionated in the same tion techniques, this essential measurement has not been made on solvents by a variant of normal phase chromatography known as many hydrophobic proteins, and so proteomic data are incomplete. hydrophilic interaction chromatography (HILIC). HILIC is per- We have extracted membrane proteins from bovine mitochondria formed with a partially aqueous mobile phase and decreasing and detergent-purified NADH:ubiquinone oxidoreductase (com- gradients of organic solvent, and so the components in the plex I) with organic solvents, fractionated the mixtures by hydro- mixture being fractionated elute in order of increasing polarity philic interaction chromatography, and measured the molecular (9). Many of the proteins recovered from the HILIC column masses of the intact membrane proteins, including those of six bind irreversibly to reverse-phase columns. Then the molecular subunits of complex I that are encoded in mitochondrial DNA. masses of the fractionated membrane proteins were determined These measurements resolve long-standing uncertainties about by electrospray MS. As part of our attempts to help characterize the interpretation of the mitochondrial genome, and they contrib- the mitochondrial membrane proteome, we have applied these techniques to membranes from bovine mitochondria. The inner ute significantly to the definition of the covalent composition of membranes contain Ͼ100 intrinsic membrane proteins involved complex I. in oxidative phosphorylation, transport of small molecules in and ͉ ͉ ͉ ͉ out of the organelle, import of nuclear encoded proteins and ion chromatography extraction complex I hydrophobic subunits channels. Many additional peripheral membrane proteins are mass spectrometry bound to the intrinsic membrane proteins, for example in the respiratory complexes. In the present work, the molecular pproximately one-third of the proteins encoded in genomes masses of Ϸ30 of these hydrophobic proteins have been mea- Aare hydrophobic membrane proteins, and yet the mass sured. The procedures were also shown to be useful for the spectrometric analysis of such proteins remains technically chal- analysis of hydrophobic proteins in the membrane-bound protein lenging. The difficulties of fractionating membrane proteins on complex NADH:ubiquinone oxidoreductase (or complex I) that 2D gels to allow their proteomic analysis are well known. Many has been purified from inner membranes of bovine mitochondria of them fail to enter the first-dimension isoelectric focusing gel in the presence of dodecylmaltoside. Bovine complex I is an and are lost. Others are not detected by staining. Nonetheless, assembly of 45 proteins with a combined molecular mass of Ϸ1 many membrane proteins have been identified by mass spectro- MDa (10, 11). They are organized into an L-shaped assembly metric analysis of peptides allowing the proteins to be associated with a peripheral arm projecting into the mitochondrial matrix with specific gene sequences, although few constituent peptides attached to an intrinsic membrane arm (12, 13). Among others, are analyzed usually and therefore the coverage of the sequence the molecular masses of six extremely hydrophobic proteins have is low. However, because of posttranslational modifications, the been measured in this way. These six proteins are all encoded in mitochondrial DNA (14). They form most of the membrane arm covalent structure of a protein is incompletely defined by its gene Ϸ ␣ sequence. Sometimes the modifications can be deduced from the of the complex by contributing 50 transmembrane -helices precise molecular mass of the protein measured by MS and (15). These measurements resolve some residual ambiguities about the interpretation of the sequence of mammalian mito- further analysis of protein ions (1, 2), but often more detailed chondrial DNA (14, 16), and also they are another significant analyses of constituent peptides are needed to identify exact sites step toward defining the precise chemical composition of of modification. To carry out intact molecular mass measure- complex I. ments on membrane proteins, they have to be removed from their phospholipid environment and kept in a soluble state while Results and Discussion being purified. The most common way of achieving this end is to Extraction of Proteins from Mitochondrial Membranes and Complex I. extract and fractionate the membrane proteins in detergent, and In previous work (17), Ϸ15–20 membrane proteins were ex- to analyze peptides in protease digests of the fractionated tracted from bovine mitochondria in mixtures of chloroform and detergent solubilized proteins by MS. Alternatively, membrane proteins have been extracted in organic solvents, fractionated by SDS͞PAGE, and identified by MS analysis of peptide digests (3, Author contributions: J.E.W. designed research; J.C. performed research; J.C. and I.M.F. 4). Unfortunately, detergents are incompatible with the MALDI analyzed data; and J.E.W. wrote the paper. and electrospray MS techniques used for measuring accurate The authors declare no conflict of interest. masses of intact proteins, and so the detergents have to be Freely available online through the PNAS open access option. removed first while the protein is kept soluble in an organic Abbreviations: HILIC, hydrophilic interaction chromatography; ESI, electrospray ionization; solvent. This objective has been achieved by chromatography in ND, subunits of NADH dehydrogenase encoded in mitochondrial DNA. 60% formic acid, allowing protein masses of detergent-purified *To whom correspondence should be addressed. E-mail: [email protected]. proteins to be measured (5). Other proteins have been purified © 2006 by The National Academy of Sciences of the USA

16170–16175 ͉ PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607719103 Downloaded by guest on September 29, 2021 Fig. 1. Extraction of membrane proteins from mitochondria and isolated complex I with organic solvent. The solvent contained 70% propan-2-ol and 25% acetonitrile, 0.56% hexafluoro-isopropanol, and aqueous buffer at various pH values (see Experimental Procedures). (A and C) Extraction of bovine mitochondria and complex I at pH 2.7, 3.7, 4.7, 6.0, and 10.8 (lanes b–f, respectively). M, molecular mass markers (kDa); lanes a, total mitochondrial proteins and complex I, respectively. (B and D) Identities of proteins in pH 3.7 extracts of mitochondria and complex I, respectively, determined by tandem MS of tryptic digests of the bands. In B and D, the positions of proteins that previously had not been characterized from either mitochondria or complex I, respectively, are shown on the left of the gel, and those of known components are shown on the right. The position of subunit ND4L was determined with an antiserum.

methanol and fractionated by gel filtration in the solvent. by tandem MS analysis of a cyanogen bromide peptide (19). However, many intrinsic membrane proteins were not in the However, subunit ND6 was not detected in either extract. extracts, and the selectivity for membrane proteins was lost when the samples contained detergents. Therefore, in the present Fractionation of Solvent Extracts by HILIC. Almost all of the proteins work, a number of solvents that might be compatible with other in the extract of mitochondrial membranes were bound to the kinds of chromatography were used to extract proteins from column. They were eluted by a linear gradient of decreasing mitochondrial membranes. Buffered mixtures of propan-2-ol organic solvent (Fig. 2A), analyzed by SDS͞PAGE (Fig. 2B), and and acetonitrile proved to be the most suitable. The optimal pH identified by tandem MS analysis of tryptic peptides (Table 4, of extraction for achieving maximal selectivity and yield of which is published as supporting information on the PNAS web membrane proteins was 3.7. At lower pH, the extracts contained site). Thus, transmembrane protein 14C was found, as well as 25 a wider range of proteins, including globular proteins (Fig. 1A). of the 29 components that had been identified by tandem MS Above pH 3.7, progressively fewer membrane proteins were analysis of the unfractionated extract. extracted, the quantities diminishing with increasing pH. The Most of the subunits extracted from complex I were retained composition of the solvent mixture was investigated also, and completely by the HILIC column, except for the ND subunits, mixtures of acetonitrile containing Ͼ60% (vol͞vol) propan-2-ol which were retained partially (Fig. 2 C and D). Minor variations provided conditions for selective extraction (Fig. 4, which is in elution positions of subunits (although not elution order) from published as supporting information on the PNAS web site). The one experiment to another were attributed to differences in the amount of detergent retained in different samples of complex I extracts were analyzed by SDS͞PAGE and tandem mass analysis by concentration of dilute solutions by membrane filtration. of tryptic digests of the stained bands (Fig. 1B). In this way, 29 proteins were identified in the extract (Table 2, which is pub- Measurement of Intact Molecular Masses. The intact molecular lished as supporting information on the PNAS web site). masses of 24 proteins in the HILIC fractions from the mito- As with mitochondrial membranes, the number of proteins chondrial extract were measured by electrospray ionization extracted from complex I and the specificity of extraction were (ESI)-MS (Table 1 and Table 5, which is published as supporting both influenced by the pH and composition of the solvent. information on the PNAS web site). Twenty-two of them were Again, the most suitable pH value was 3.7 (Fig. 1C). From the identified also in the unfractionated extract, but two others, 45 different subunits that form the complex, 22 were detected in complex III subunit XI and cytochrome oxidase subunit VIII, the extract (see Fig. 1D and Table 3, which is published as had not been found in the unfractionated extracts. Therefore, the supporting information on the PNAS web site). All of them, total number of proteins that were found in the extract was 33 except subunits B14.5a, B14, B13, B8, and 10 kDa, are authentic (29 plus ND4L in the unfractionated material, plus transmem- membrane proteins with the potential to form at least one brane protein 14C, subunit XI of complex III, and subunit VIII transmembrane ␣-helix. Fifteen of them have been characterized of cytochrome oxidase). Thirty-one of them seem to be authentic before (18, 19). As in the mitochondrial extract, 6 of the 7 membrane proteins with the capacity to form at least one hydrophobic ND subunits that are encoded in mitochondrial transmembrane ␣-helix. The exceptions are subunit IX of com- DNA were found. Subunit ND4L was detected in both extracts plex III [which is the mitochondrial import sequence cleaved with an antibody (see Fig. 5 and Supporting Experimental Pro- from the Rieske protein that remains associated with the com- cedures, which are published as supporting information on the plex III outside the membrane domain (20)] and cytochrome PNAS web site). Not only is ND4L hydrophobic, it contains only oxidase subunit VIb.

a single tryptic cleavage site, and so it has not been detected by The intact molecular masses of 18 of 24 of the proteins have BIOCHEMISTRY mass mapping of tryptic digests, although it has been identified been characterized before (Table 5) (2, 18, 19, 21–24). However,

Carroll et al. PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 ͉ 16171 Downloaded by guest on September 29, 2021 Fig. 2. Fractionation of hydrophobic proteins by HILIC. Solvent extracts of bovine mitochondria and complex I were made at pH 3.7 and fractionated by HILIC. (A and C) Elution profiles (solid lines) monitored by UV absorbance at 225 nm. The column was equilibrated in buffer 1 and eluted with a gradient (dotted lines in A and C) of buffer 2. The flow rates in A and C were 0.1 and 0.05 ml͞min, respectively. For additional details, see Experimental Procedures.(B and D) Analysis of fractions by SDS͞PAGE. Lanes Ex, extracts of mitochondria and complex I, respectively; M, molecular mass markers. The identified proteins are indicated on the right with fraction numbers and peak names in parentheses.

the intact masses of six of the proteins had not been measured and of brain protein 44-like are serine and alanine, respectively, previously (summarized in Table 1), although they have been the two most common acetylated N-terminal residues (30), and identified as components of mitochondria (25, 26). One of them, the N terminus of mature transmembrane protein 14C is proline. ND2, is encoded in mitochondrial DNA, and five others are Five additional subunits of complex I that are encoded in encoded in the nucleus. These data show that, in common with mitochondrial DNA (ND1, ND3, ND4, ND4L, and ND5) were other proteins encoded in mtDNA (17) and with proteins detected in the mitochondrial extract (Fig. 1B), but there was encoded in chloroplast genomes (5, 27), ND2 has retained its insufficient material to permit measurement of their intact translational initiator, formylmethionine, at residue 1, and that masses. However, by fractionation of the extract of complex I, it is not modified at any other site. These data demonstrate also the same subunits plus subunit ND2 were recovered as simplified that the translational initiator methionine residues of four of the mixtures in the break-through of the HILIC column in sufficient five nuclear-encoded subunits (transmembrane protein 14C, quantity to allow their molecular masses to be measured (see Fig. Usmg5, brain protein 44, and brain protein 44-like) have been 2). The spectra show that all six subunits have retained their removed. Two of them (transmembrane protein 14C and translational initiator formylmethionine and that otherwise they Usmg5) are not modified further, whereas brain protein 44 and are unmodified (Table 1 and Fig. 3). brain protein 44-like have been acetylated, probably at their N Tandem mass analysis of the N-terminal peptide of subunit termini. In the remaining nuclear encoded subunit, phospho- B14.7 of complex I has shown previously that the translational lamban, the translational initiator methionine has been retained initiator methionine has been removed, and that alanine-2 is and N-␣-acetylated (Fig. 6B, which is published as supporting acetylated (31). The intact subunit has never been recovered by information on the PNAS web site). Phospholamban is a com- reverse-phase HPLC fractionation of subunits of complex I (18, ponent of the sarcoplasmic reticulum (28), although it has been 19), but it was obtained by HILIC fractionation of the extract of detected before in mitochondria (26, 29), and so it may be complex I, allowing its intact molecular mass to be measured targeted to both compartments. Residue 2 of brain protein 44 (Table 1 and Fig. 7, which is published as supporting information

16172 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607719103 Carroll et al. Downloaded by guest on September 29, 2021 Table 1. Molecular masses of some hydrophobic proteins extracted from bovine heart mitochondria and complex I Mass, Da

Protein Observed Calculated Mass difference Modification

Transmembrane protein 14C* 11,604.4 11,735.0 Ϫ130.6 ϪMet Usmg5*† 6,303.5 6,434.6 Ϫ131.1 ϪMet Brain protein 44-like* 12,299.5 12,388.6 Ϫ89.1 ϪMet ϩ acetyl Brain protein 44* 14,193.1 14,281.9 Ϫ88.8 ϪMet ϩ acetyl Phospholamban* 6,122.7 6,080.5 ϩ42.2 ϩ acetyl ND1‡ 35,699.1 35,670.2 ϩ28.9 ϩ formyl ND2*‡ 39,283.5 39,254.4 ϩ29.1 ϩ formyl ND3‡ 13,082.4 13,054.7 ϩ27.7 ϩ formyl ND4‡ 52,130.0 52,099.4 ϩ30.6 ϩ formyl ND4L‡ 10,825.4 10,797.3 ϩ28.1 ϩ formyl ND5‡ 68,319.8 68,286.8 ϩ33.0 ϩ formyl B14.7‡ 14,667.5 14,758.1 Ϫ90.6 ϪMet ϩ acetyl

Masses were measured by ESI-MS and calculated from protein sequences. The masses of the proteins in the table have not been measured previously. The masses of other proteins in the mixtures that confirm known values are summarized in Tables 5 and 6. *Isolated from bovine mitochondrial membranes. †Up-regulated during skeletal muscle growth protein 5, also known as diabetes-associated protein in insulin- sensitive tissue (DAPIT). ‡Isolated from bovine complex I.

on the PNAS web site). This experiment confirmed the N-␣- procedure is rather selective for membrane proteins, but for acetylation of residue 2 as its only posttranslational modification. reasons that are as yet obscure, not all hydrophobic protein It is the most hydrophobic of the nuclear encoded proteins of the components of the mitochondrial membranes are present in the complex, with the potential to form three transmembrane ␣-he- extracts. For example, two notable absentees are the c-subunit of lices. The ESI-MS analysis of the HILIC fractions also confirmed ATP synthase and the ND6 subunit of complex I, which are both the molecular masses of 11 subunits of bovine complex I that had abundant components of the membranes. Also, it has the been characterized before (Table 6, which is published as advantages of allowing membrane proteins to be extracted from supporting information on the PNAS web site). detergent-purified protein complexes and of being compatible with HILIC. The use of this form of chromatography for Biological Significance. The development of methods to permit the purifying membrane proteins is a new development that has fractionation and characterization of membrane proteins by MS allowed some well known, but hitherto poorly characterized has a general significance for proteomic studies. The extraction proteins to be studied, namely six of the seven hydrophobic

Fig. 3. Analysis by ESI-MS of subunits ND1, ND2, ND3, ND4, ND4L, and ND5 of complex I. (A and C) Analysis of fractions FT2 and FT1, respectively, from Fig. 2C. (A) The four multiply charged series (components A–D) correspond to subunits ND2, ND1, ND4, and ND5, respectively. (C) The two multiply charged series

(components A and B) correspond to subunits ND3 and ND4L, respectively. (B and D) The spectra, reconstructed on a true molecular mass scale, show the molecular BIOCHEMISTRY masses (in daltons) of the subunits (summarized in Table 1).

Carroll et al. PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 ͉ 16173 Downloaded by guest on September 29, 2021 proteins that form most of the membrane arm of complex I and SDS͞PAGE Analysis of Proteins. Mitochondrial extracts (Ϸ100 ␮l) are encoded in mammalian mitochondrial DNA. were delipidated by addition of 4 vol of diethyl ether at Ϫ20°C. The measurement of the molecular masses of subunits ND4, After being kept at Ϫ20°C for 30 min, the samples were ND4L, and ND5 helps to remove some long-standing uncer- centrifuged (16,000 ϫ g, 10 min, 4°C). The pellets were resus- tainties about the interpretation of the sequences of bovine and pended in 100 ␮l of a mixture of chloroform and methanol (2:1, human mitochondrial genomes (14, 16). The molecular masses vol͞vol) containing 1 mM DTT and 0.01 vol of a 20% (wt͞vol) of bovine subunits ND4 and ND4L show that the corresponding solution of SDS. SDS (20%) was added also to the same do overlap by 7 bases, as proposed but never proven, and concentration to portions of organic solvent extracts of complex the molecular mass of ND5 is compatible with the proposed I. The samples were dried in vacuo and resolubilized in sample 17-base overlap of the corresponding gene with that of ND6 on buffer, and the pH values of any acidic samples were adjusted to the opposite DNA strand. The measured mass of ND4 corre- neutrality. Samples were analyzed by SDS͞PAGE on 12–22% sponds to the product from the polyadenylated ND4 mRNA gradient gels and stained with Coomassie Blue R250 (41). rather than to that of ND4X, the proposed ‘‘extended’’ version of the protein that could be made by read-through from ND4 Protein Identification. Excised protein bands from SDS͞PAGE into the following tRNAHis gene, producing a protein 17 aa gels were digested with trypsin (Roche, Mannheim, Germany) longer. It now seems less likely that this read-through mechanism (31, 42). Peptides were extracted from the gel in 4% formic acid operates in either bovine or human mitochondria. containing 60% acetonitrile. Alternatively, proteins in portions The molecular masses of the ND subunits (excepting ND6) of HILIC fractions were dried completely in vacuo, redissolved show that, apart from the retention of their N-terminal formyl in 50 mM ammonium bicarbonate containing 0.25 mM CaCl2, groups, the proteins are not covalently modified. This finding is and digested with trypsin at 37°C. Peptides were analyzed by significant in the context of the proton-pumping mechanism of peptide mass fingerprinting and tandem MS peptide sequencing complex I. Transfer of two electrons, one at a time, from NADH in a 4700 MALDI-TOF-TOF mass spectrometer (Applied Bio- to ubiquinone, via flavin mononucleotide and nine iron-sulfur systems, Warrington, U.K.) by using ␣-cyano-4-hydroxy- clusters, is accompanied by the transfer of four protons from the transcinnamic acid as the matrix. Sample data were calibrated matrix of the to the inter-membrane space (32). internally with the autolysis products of trypsin. Monoisotopic However, the mechanism of proton transfer is not understood. peak mass lists and tandem MS data were screened against Various proposals have been made, including a conformational NCBInr protein sequence databases by using MASCOT from coupling mechanism (33–35), a mechanism related to the Q- either an internal server or via a web interface (43). Alterna- cycle that operates in complex III (36), and a mechanism tively, tandem MS analysis was performed in a Q-TOF instru- depending on the possible presence of an unidentified cofactor ment (Micromass, Altrincham, U.K.) as described (41). in the membrane domain of the enzyme (37, 38). If this cofactor exists, it is not attached to any of the six ND subunits that have Fractionation of Extracts. Protein extracts of mitochondrial mem- been analyzed (assuming that it is stable at pH 3.7), nor to any branes and complex I were fractionated on polyhydroxyethyl- of the 13 nuclear-encoded subunits that contribute to the aspartamide columns (100 mm ϫ 2.1 mm i.d. or 100 mm ϫ 1mm membrane domain. It remains possible that such a cofactor i.d, 300-Å pore size, and 5-␮m particle size; PolyLC, Columbia, could be attached to ND6, or that it could be bound nonco- MD) equilibrated in buffer 1 (63% propan-2-ol͞22.5% aceto- valently to the membrane arm of complex I. nitrile͞0.5% hexafluoro-isopropanol͞14.0% water͞20 mM am- monium formate, pH 3.7). The proteins were eluted with a linear Experimental Procedures gradient of buffer 1 with buffer 2 (30% propan-2-ol͞0.5% Preparation of Bovine Heart Mitochondria and Purification of Complex I. hexafluoro-isopropanol͞69.5% water͞20 mM ammonium for- Mitochondria were isolated from bovine hearts, and complex I mate, pH 3.7). The absorbance of the eluate was monitored at was purified from them in the presence of n-dodecyl-␤-D- 225 nm. maltoside (Anatrace, Maumee, OH) as described before (19, 39, 40). The purified complex I was concentrated to Ϸ5–7 mg͞ml on Molecular Mass Measurements. The protein contents of fractions a Vivaspin-4 membrane with a molecular mass cut-off of 100 kDa were analyzed by LC-ESI-MS ‘‘on-line’’ to the column (50 ϫ 1 (Sartorius, Go¨ttingen, Germany), and the concentrate was mm i.d.) with a Quattro Ultima triple quadrupole mass spec- stored in liquid nitrogen. trometer (Micromass). Alternatively, samples were introduced ‘‘off-line’’ into a stream (flow rate 2–3 ␮l͞min) of 50% aqueous Extraction of Membrane Proteins from Mitochondria and Complex I. acetonitrile into either an electrospray interface of a Quattro Mitochondria were resuspended at 4°C in a solution consisting Ultima instrument, or a nano-flow electrospray interface of a of 2 mM Tris⅐HCl (pH 7.4), 250 mM sucrose, and 1 mM EDTA Q-TOF mass spectrometer (Micromass). In some cases, the and then centrifuged (16,000 ϫ g, 6 min, 4°C). The pellet spectra were improved by addition to the sample of formic acid (defined as 1 vol), or the sample of complex I, was vortexed (final concentration 1–5%, vol͞vol). Both mass spectrometers occasionally for 5 min at room temperature in 9 vol of mixtures were operated in positive ion single MS mode. They were of propan-2-ol and acetonitrile from 95% (vol͞vol) propan-2-ol calibrated with horse heart myoglobin and bovine trypsinogen. to 95% (vol͞vol) acetonitrile containing 0.56% hexafluoroiso- Protein molecular masses were determined from the series of propanol and 4.44% water buffered to a final concentration of multiply charged ions by using the component analysis function 20 mM with formic acid (pH 2.7), ammonium formate (pH 3.7 of MassLynx software, version 3.5 (Micromass). and 4.7), ammonium acetate (pH 6.0), or ammonium hydroxide (pH 10.8). Insoluble material was removed by centrifugation We thank Dr. Richard Shannon (Medical Research Council, Cambridge, (16,000 ϫ g, 10 min, 18°C). U.K.) for samples of bovine mitochondrial complex I.

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