GRIM-19, a Cell Death Regulatory Gene Product, Is a Subunit of Bovine Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I)*

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Iλ, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-β and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) catalyzes the first step in the electron transport chain, the oxidation of NADH to NAD ϩ coupled to proton translocation across the inner mitochondrial membrane (1)(2)(3)(4). Complex I from bovine heart mitochondria is a highly complex, multisubunit, membrane-bound assembly, with a molecular mass of over 900 kDa. The sequences of 42 of the subunits of complex I have been reported previously (3,(5)(6)(7); seven are mitochondrial gene products, whereas the rest are encoded in the nucleus and imported into the organelle from the cytoplasm. Prokaryotic complex I systems are simpler assemblies of 13-14 subunits, all of them conserved in eukaryotes, and contain an equivalent set of redox cofactors (8,9). Mitochondrial complex I is a roughly L-shaped assembly (10 -12), with one arm in the plane of the lipid bilayer. The other, containing the more hydrophilic subunits, protrudes from the membrane and can be dissociated from it, producing subcomplex I (13,14). The oxidation of NADH is catalyzed at an active site containing non-covalentlybound flavin mononucleotide, and electrons are transported through the assembly to the ubiquinone acceptor via ironsulfur clusters. Subcomplex I contains 15 subunits of complex I and all of its known redox cofactors but has no bound ubiquinone; it is thus active in NADH oxidation but only when coupled to artificial electron acceptors such as ferricyanide or hexaamineruthenium.
With the aim of unambiguously defining the subunit composition of subcomplex I (13,14), each of the bands resolved by SDS-PAGE 1 analysis was examined by peptide-mass mapping. In this way, 14 known subunits of mitochondrial complex I were confirmed as being present in the subcomplex. In addition, a 15th band, not corresponding to any known subunit, was observed, running close to subunit B17.2. This additional protein, which is tightly associated with subcomplex I, was investigated by mass mapping and tandem MS of its tryptic peptides, and the corresponding cDNA was sequenced. The mature protein, of 143 amino acids, has an acetylated ␣-amino group. The presence of the new protein has also been confirmed in intact complex I, and following the nomenclature for mitochondrial complex I it has been named B16.6 (3). The amino acid sequence clearly identifies subunit B16.6 of mitochondrial complex I to be the bovine homolog of the GRIM-19 protein, the product of a cell death regulatory gene induced by interferon-␤ and retinoic acid. GRIM-19 has previously been detected in HeLa cells, predominantly in the nucleus, though punctate staining of the cytoplasm was also observed (15,16).

EXPERIMENTAL PROCEDURES
Isolation of Bovine Mitochondrial Complex I and the I Subcomplex-Mitochondria were isolated from bovine hearts (17), and mitochondrial membranes were prepared by disruption with a Waring blender in the presence of potassium chloride (18). Complex I was purified according to Sazanov et al. (14), by solubilization with dodecylmaltoside (Anatrace, Maumee, OH) and ion-exchange chromatography on Q-Sepharose HP media (Amersham Pharmacia Biotech). Pooled complex I fractions were either used to prepare subcomplex I (see below) or subjected to further purification using a second ion-exchange separation, ammonium sulfate precipitation, and gel filtration (14). All purification steps were carried out at 0 -4°C. Subcomplex I was prepared by a procedure developed from that of Finel et al. (13). Complex I, from the first ion-exchange column above, was precipitated by addition of dodecylmaltoside to 1%, sodium cholate (Dojindo Laboratories, Kumamoto, Japan) to 1.8%, and ammonium sulfate to 50% saturation. After centrifugation, the resulting pellet was dissolved into 1% LDAO (Calbiochem), 100 mM potassium phosphate, pH 7.5, and 1 mM dithiothreitol (Melford Laboratories, Ipswich, UK), to a final concentration of ϳ10 mg ml Ϫ1 . Portions were layered onto linear sucrose gradients (12 ml, 5-15% sucrose, 0.5% LDAO, 500 mM potas-* This work was supported by the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  sium phosphate, pH 7.5, 1 mM dithiothreitol) and centrifuged for 18 h at 200,000 ϫ g. The sharp yellow-brown band in the center of the gradient was concentrated to ϳ6 mg ml Ϫ1 by ultrafiltration (YM-100; Millipore, Bedford, MA), and portions (0.5 ml) were injected onto a Superose 6 HR 10/30 gel filtration column (Amersham Pharmacia Biotech) and eluted (0.3 ml min Ϫ1 ) in a buffer containing 0.1% LDAO, 100 mM potassium phosphate, pH 7.5, and 1 mM dithiothreitol. The colored fractions spanning the apex of the symmetrical absorbance peak (280 nm) were pooled and stored in liquid nitrogen (concentration ϳ1 mg ml Ϫ1 ).
Protein concentrations were determined by the BCA protein assay (Pierce) using bovine serum albumin as standard. Unless otherwise stated, all chemicals were purchased from Merck and were of analytical grade.
Mass Spectrometry-Proteins separated by SDS-PAGE were identified by peptide mass fingerprinting. Bands were excised from the gel, cleaved "in-gel" with trypsin (21), and the mixture of tryptic peptides was analyzed by MALDI-TOF MS using a TofSpec 2E mass spectrometer (Micromass, Altrincham, UK) and ␣-cyano-4-hydroxy-trans-cinnamic acid as the matrix. Spectra were calibrated using peptides from trypsin autolysis at 2163.057 and 2273.160, and a matrix-related ion at 1060.048 was present in each spectrum.
Peptide sequences were obtained by tandem MS after CID with argon gas in a Q-TOF mass spectrometer equipped with ESI (Micromass Q-TOF; Altrincham, UK), which was tuned and calibrated using the CID fragments of a synthetic peptide, [Glu 1 ] fibrinopeptide B (Sigma-Aldrich). Peptide mixtures from in-gel tryptic digest were desalted on a Poros R2 50-Å reverse-phase support (PerSeptive Biosystems), eluted in 1 l of 1% formic acid, 60% methanol, introduced into the mass spectrometer from a nano-electrospray interface (22), or transferred directly (1 l min Ϫ1 ) from an "on-line" capillary HPLC (CapLC; Micromass), following fractionation on a PepMapC18 column (LC Packings, Amsterdam, The Netherlands) with an acetonitrile gradient in 0.1% formic acid. MS and tandem MS data were acquired automatically.
Peptide fragmentation spectra were interpreted manually, assembled into Peptide Sequence Tags (23), and compared with protein sequence data bases. Peptide sequences were also used to design synthetic oligonucleotide primers (see below) and later for the verification of the cDNA-derived sequence (see Table I).
Subunit B16.6 was also purified by reverse-phase HPLC of subcomplex I on an Aquapore RP-300 column (PerkinElmer Life Sciences), with an acetonitrile gradient in 0.1% trifluoroacetic acid. Before application to the column, samples were mixed with an equal volume of 6 M guanidine hydrochloride in 0.1% trifluoroacetic acid and then centrifuged. Subunit B16.6 eluted at ϳ63% acetonitrile, immediately following subunit PSST and often co-eluting with the 30-kDa subunit. Portions of eluate were examined by ESI MS as described below. Tryptic digests of the purified subunits, analyzed by tandem MS, provided additional, confirmatory sequence data (see Table I).
The molecular mass of intact B16.6, purified by reverse-phase HPLC, was measured by ESI MS using a Sciex API III ϩ triple quadrupole mass spectrometer, which was tuned and calibrated with a mixture of poly(propylene glycols) over the range of m/z 59 to 2010 and checked using horse heart myoglobin (average mass 16951.4 Da). Samples were introduced, via a Rheodyne loop, into a stream (3 l min Ϫ1 ) of 50% aqueous acetonitrile, and spectra were recorded by scanning the first quadrupole (Q1) from 700 to 1700 m/z.
Amplification and Sequencing of Bovine cDNA-cDNA was amplified from total bovine heart cDNA by PCR using Expand DNA polymerase (Roche Diagnostics Gmbh, Mannheim, Germany). First, the mixed primers F1, F2, R1, and R2, based on partial amino acid sequences from tandem MS, were synthesized (see Fig. 2A). Primers F1 (CARGAYAT-GCCNCCNGT) and R1 (GTRTACCANARRAANCC) were used to amplify a partial cDNA, and the product of this reaction was then used as template for a second reaction with the two nested primers F2 (ATH-GARGAYTTYGARGC) and R2 (CCRTANARYTCNCCCAT). The PCR product from the second reaction was sequenced directly. The partial sequence was extended to the 5Ј-and 3Ј-ends using unique primers. For the 3Ј-extension, the primer F3 (see Fig. 2A) and a reverse oligo(dT) primer were used, and the PCR product was sequenced directly. For the first 5Ј-extension, bovine heart cDNA with a 5Ј-poly(A) tail was used as template with reverse primer R3, and the PCR product was sequenced directly, thereby extending the sequence to base 145 (see Fig. 2A). In a second 5Ј-extension reaction, bovine heart cDNA synthesized using a SMART PCR cDNA synthesis kit (CLONTECH, Palo Alto, CA) was used as a template, with the forward primer TAGAAGCTTGAATTCG-GATCCCGCAGAGTACGCGGG (F4) and reverse primer R4. The product was cloned into a pET vector, and colonies identified by PCR screening were used for sequencing. This yielded a set of clones of different lengths, the longest extending 31 bases upstream of the ATG initiator codon. All the cDNA sequences reported, excepting bases 1-3, have been confirmed by at least two independent DNA sequences.

Detection of a New Subunit in Bovine Mitochondrial Complex
I and Subcomplex I-The subunits of bovine mitochondrial complex I and subcomplex I were separated by SDS-PAGE ( Fig. 1) and analyzed by peptide mass fingerprinting (data not shown). This confirmed the presence of 14 known components of complex I in subcomplex I (Fig. 1) and also revealed a further band that did not correspond to any known complex I subunit. The new protein was also identified in intact complex I. It could not be removed from the complex by any of the following procedures: extended washing of complex I bound to an ion-exchange column, gel filtration, dialysis, treatment with 2 M NaCl followed by dialysis, ammonium sulfate precipitation, or precipitation by dialysis against a low ionic strength buffer. In addition, the new protein is also present in subcomplex I, prepared by disrupting complex I with LDAO, and purified using a sucrose gradient and gel filtration. Therefore, the new protein is not adventitiously bound to complex I, and because it is present in the complex at approximately the same molar ratio as other subunits (as estimated by gel band intensities and the relative yields in HPLC experiments) it is reasonable to conclude that it is a bona fide subunit of the assembly. The new protein gave no N-terminal sequence by Edman degradation, suggesting that the N terminus is modified (see below) and in accordance with the nomenclature for bovine mitochondrial complex I has been named B16.6 (3). Subunit B16.6 is observed reproducibly in all our preparations of bovine complex I and subcomplex I. The complicated SDS-PAGE patterns of complex I and the inaccessibility of the N terminus provide likely reasons why B16.6 has been overlooked previously.  (12-22%), and the identities of subunits present in subcomplex I, shown at the side, were confirmed by peptide-mass mapping. All subunits found in subcomplex I are also present in complex I, which also contains 28 additional subunits. Also marked is the band corresponding to the new subunit, which runs close to B17.2.

GRIM-19 Is a Subunit of Mitochondrial Complex I 38346
Tandem Mass Spectrometry-The tryptic peptide mixture from the unidentified protein band was examined by tandem MS, and amino acid sequences from several tryptic peptides were determined (Table I) (Table I) also aligned readily with the human sequence, suggesting B16.6 to be a homolog of human GRIM-19; they later confirmed the amino acid sequence deduced from cDNA sequencing (see below).   Identified sequences within peptides are underlined (I and L are isobaric and are not distinguishable). Peptides T2-3, T2-4, and T13-15 show  artefactual mass differences because of deamidation, and peptide T21 is sodiated. Peptides T2-3, T2-4, T3, T11-12, T12, T13, T19, and, additionally, T3-4 (residues 7-22) and T20 (115-127) were also detected by MALDI-TOF MS.  Fig. 2A. The 5Ј-sequence of the cDNA extends 31 bases upstream of the proposed initiator methionine codon and does not contain an in-phase stop codon. The mass of the intact protein measured by ESI MS (16585 Da) was found to be consistent with the mass calculated from the sequence (16584.3 Da), provided that the initiator methionine is cleaved from the mature protein and that the N-terminal alanine residue is acetylated.
The B16.6 subunit has a predicted isopotential point of 9.7. Values above pH 9.5 are predicted for approximately half the subunits of mitochondrial complex I and for almost all of the 12 nuclear-encoded subunits with modified N termini. The hydropathy profile (not shown) demonstrates the amphipathic nature of subunit B16.6 (24), with one potential transmembrane ␣-helical domain between residues 29 and 47 (25). These properties are consistent with B16.6 comprising part of subcomplex I; whereas it is expected to be predominantly hydrophilic, isolated subcomplex I aggregates in the absence of detergent, suggesting the presence of hydrophobic surface patches. Because of its strong association with subcomplex I, subunit B16.6 is likely to be located predominantly on the matrix side of the inner mitochondrial membrane, but its topography in the inner membrane is not known, and either residues 1-28 or 48 -143 could lie in the intermembrane space.
Identification of the New Complex I Subunit, B16.6, as GRIM-19 -The sequences of human GRIM-19 (16) and B16.6 from bovine complex I are 83% identical (Fig. 2B). Data base entries (EMBL and EST) for other vertebrate homologs also show high identities with the bovine protein: 94, 83, 81, 73, 73, and 70% for pig, mouse, rat, zebrafish, oryzias, and chicken, respectively. In all species (see also Fig. 2B), the N-terminal sequences are highly conserved, though deletion of the N-terminal 50 amino acids of GRIM-19 did not inhibit cell death (16). The 25 C-terminal amino acids are markedly less conserved. In addition, there are a number of plant homologs, for example from soybean, sorghum, cotton fiber, wheat, barley, and barrel medic, which show high identity with each other but a maximum of 46% identity with bovine B16.6. However, the sequence of residues 97-109 remains highly conserved. Deletion of the C-terminal 43 amino acids of GRIM-19 showed their importance in promoting cell death (16), consistent with an important role for residues 97-109. However, this motif does not appear to have the characteristics of an ATP binding domain (26) as was suggested by Angell et al. (16).
Post-translational Modification and Targeting of B16.6 to Mitochondria-The acetylation of the alanine N terminus and the presence of a methionine at position Ϫ1 indicate that, in common with 18 other subunits of complex I, no cleavable presequence is used to target B16.6 to mitochondria (3,6). Of these 18 subunits, 12 are modified at the N terminus, 11 by acetylation and 1 by myristylation; for such proteins, the localization signal must lie within the mature protein (27,28). The cellular distribution of GRIM-19 (B16.6) is complex. It has been detected, with antibodies, primarily in the nucleus of HeLa cells, though "punctate" staining in the cytoplasm, possibly corresponding to mitochondria, was also observed (16). Furthermore, transcripts of GRIM-19 are particularly elevated in human heart and skeletal muscle and to a lesser extent in liver, kidney, and placenta, correlating approximately with the relative abundance of mitochondria in these tissues (16). Although only one human gene for GRIM-19 has been detected (29), it remains possible that differential splicing provides the means to target the protein to more than one site. A second possibility is that targeting is controlled by a post-translational modification such as acetylation or phosphorylation. Angell et al. (16) have suggested that human GRIM-19 is phosphorylated constitutively, but there is no indication from our mass spectrometric analysis for a phosphoryl group in mitochondrial B16.6. Another mitochondrial protein, endonuclease G, has been shown to be released from mitochondria during apoptosis and subsequently translocated to the nucleus (30). Clearly the targeting of GRIM-19 (B16.6) to either nucleus or mitochondria requires further study.
Concluding Remarks-B16.6 is the 43rd subunit of bovine mitochondrial complex I to be identified. It is possible that it represents the completion of the primary structure of this complex enzyme, although a protein with a molecular mass of 10566 (Ϯ2) Da, observed in some preparations, has not yet been explained (5).
The GRIM-19 protein has now been found to fulfill two roles within the cell. The first is as part of the interferon-␤-and retinoic acid-induced pathway of cell death (15,16). The second is as part of the mitochondrial complex I assembly. These two seemingly disparate functions may be linked by the involvement of mitochondria in apoptotic cell death.