Joe Carroll

Bovine Complex I Is a Complex of 45 Different Subunits

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Iβ, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

Analysis of the Subunit Composition of Complex I from Bovine Heart Mitochondria

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.

The nuclear encoded subunits of complex I from bovine heart mitochondria.

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, I alpha, I beta and I lambda, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.

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.

Surface plasmon resonance analysis at a supported lipid monolayer

Methods for the formation of supported lipid monolayers on top of a hydrophobic self assembled monolayer in a surface plasmon resonance instrument are described. Small unilamellar vesicles absorb spontaneously to the surface of the hydrophobic self-assembled monolayer to form a surface which resembles the surface of a cellular membrane. Lipophilic ligands, such as small acylated peptides or glycosylphosphatidylinositol-anchored proteins, were inserted into the absorbed lipid and binding of analytes to these ligands was analysed by surface plasmon resonance. Conditions for the formation of lipid monolayers have been optimised with respect to lipid type, chemical and buffer compatibility, ligand stability and reproducibility.

A cytolytic δ-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal δ‐endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation‐selective channels in the bilayers, permeable to K+ and Na+ but not to N‐methylglucamine or Cl−, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid‐osmotic lysis.

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

The covalent structure of a protein is incompletely defined by its gene sequence, and mass spectrometric analysis of the intact protein is needed to detect the presence of any posttranslational modifications. Because most membrane proteins are purified in detergents that are incompatible with mass spectrometric ionization techniques, this essential measurement has not been made on many hydrophobic proteins, and so proteomic data are incomplete. We have extracted membrane proteins from bovine mitochondria and detergent-purified NADH:ubiquinone oxidoreductase (complex I) with organic solvents, fractionated the mixtures by hydrophilic interaction chromatography, and measured the molecular masses of the intact membrane proteins, including those of six subunits of complex I that are encoded in mitochondrial DNA. These measurements resolve long-standing uncertainties about the interpretation of the mitochondrial genome, and they contribute significantly to the definition of the covalent composition of complex I.

Assembly factors for the membrane arm of human complex I

Significance Mammalian complex I, the largest and most complicated enzyme of the mitochondrial respiratory chain, is an L-shaped assembly of 44 proteins with one arm in the mitochondrial matrix and the orthogonal arm buried in the inner membrane. It is put together from preassembled modules. This investigation concerns the little studied process of the assembly of the membrane arm module from proteins emanating from both nuclear and mitochondrial genomes. We have identified two membrane protein assembly factors C3orf1 and TMEM126B, not found in the mature complex, that help this process by putting together two membrane arm subcomplexes. Defects in the assembly of complex I are increasingly being associated with human pathologies. Mitochondrial respiratory complex I is a product of both the nuclear and mitochondrial genomes. The integration of seven subunits encoded in mitochondrial DNA into the inner membrane, their association with 14 nuclear-encoded membrane subunits, the construction of the extrinsic arm from 23 additional nuclear-encoded proteins, iron–sulfur clusters, and flavin mononucleotide cofactor require the participation of assembly factors. Some are intrinsic to the complex, whereas others participate transiently. The suppression of the expression of the NDUFA11 subunit of complex I disrupted the assembly of the complex, and subcomplexes with masses of 550 and 815 kDa accumulated. Eight of the known extrinsic assembly factors plus a hydrophobic protein, C3orf1, were associated with the subcomplexes. The characteristics of C3orf1, of another assembly factor, TMEM126B, and of NDUFA11 suggest that they all participate in constructing the membrane arm of complex I.

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

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-α-acetylated, and a 14th, subunit B18, is N-α-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-α-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

Significance Cellular powerhouses called mitochondria generate fuel known as adenosine triphosphate, or ATP, to sustain complex life. The capacity of mitochondria to do so depends on a supply of energy from oxidation of energy-rich compounds in foodstuffs to generate a chemical potential difference for hydrogen ions, called the proton motive force (pmf), across the inner mitochondrial membrane. Disruption of this membrane dissipates the pmf, and the cells die for lack of fuel. This event happens, for example, when the concentration of calcium ions inside human mitochondria is increased. The mitochondria respond by opening a pore, water enters, and the mitochondria swell and burst. The molecular identity of the pore is disputed, and we have disproved one proposal. 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.