FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2076055995> ?p ?o ?g. }

• W2076055995 endingPage "6435" @default.
• W2076055995 startingPage "6425" @default.
• W2076055995 abstract "Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear-encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNA interference had shortened lifespans and severe defects in mitochondrial respiratory chain function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in the amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the electron transport chain can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect. Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear-encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNA interference had shortened lifespans and severe defects in mitochondrial respiratory chain function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in the amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the electron transport chain can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect. The mitochondrial respiratory chain (MRC) 2The abbreviations used are: MRC, mitochondrial respiratory chain; COX, cytochrome c oxidase; RNAi, RNA interference; qRT-PCR, quantitative reverse transcriptase PCR; IGA, in-gel activity; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; CIV-IGA, complex IV in-gel activity; CI-IGA, complex I in-gel activity; ETC, electron transport chain; BNG, blue native gel; NFR, NADH-ferricyanide oxidoreductase. consists of five multisubunit complexes termed complexes I through V. The physical organization of these complexes is controversial. Two extreme models of their structure have been proposed. A “liquid-state model” of the mitochondrial respiratory chain depicts the respiratory complexes embedded in the inner mitochondrial membrane as separate entities, functionally connected to each other by the mobile electron carriers, coenzyme Q and cytochrome c. This model postulates that random collision among all respiratory components can account for measured electron transport rates in the inner mitochondrial membrane. Lateral diffusion of each component is regarded as sufficient to generate contact between MRC components (1Chazotte B. Hackenbrock C.R. J. Biol. Chem. 1988; 263: 14359-14367Abstract Full Text PDF PubMed Google Scholar, 2Hackenbrock C.R. Chazotte B. Gupte S.S. J. Bioenerg. Biomembr. 1986; 18: 331-368Crossref PubMed Scopus (289) Google Scholar). However, data also exist to support a “solid-state” model of respiratory complexes. This model proposes that mitochondrial respiratory complexes are organized into very large supercomplexes (3Chance B. Williams G.R. Nature. 1955; 176: 250-254Crossref PubMed Scopus (202) Google Scholar, 4Stroh A. Anderka O. Pfeiffer K. Yagi T. Finel M. Ludwig B. Schagger H. J. Biol. Chem. 2004; 279: 5000-5007Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 5Schagger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1040) Google Scholar, 6Eubel H. Jansch L. Braun H.P. Plant Physiol. 2003; 133: 274-286Crossref PubMed Scopus (280) Google Scholar, 7Schagger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 8Krause F. Reifschneider N.H. Goto S. Dencher N.A. Biochem. Biophys. Res. Commun. 2005; 329: 583-590Crossref PubMed Scopus (98) Google Scholar). Stoichiometric architectures of supercomplexes have been identified in multiple organisms, from prokaryotes to humans, and include I1III2, I1III2IV1-4, and III2IV4 (5Schagger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1040) Google Scholar, 7Schagger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar, 9Schagger H. IUBMB Life. 2001; 52: 119-128Crossref PubMed Scopus (166) Google Scholar, 10Schafer E. Seelert H. Reifschneider N.H. Krause F. Dencher N.A. Vonck J. J. Biol. Chem. 2006; 281: 15370-15375Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 11D'Aurelio M. Gajewski C.D. Lenaz G. Manfredi G. Hum. Mol. Genet. 2006; 15: 2157-2169Crossref PubMed Scopus (104) Google Scholar, 12Cruciat C.M. Brunner S. Baumann F. Neupert W. Stuart R.A. J. Biol. Chem. 2000; 275: 18093-18098Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Vonck J. Schafer E. Biochim. Biophys. Acta. 2008; 1793: 117-125Crossref PubMed Scopus (131) Google Scholar, 14Bianchi C. Genova M.L. Parenti Castelli G. Lenaz G. J. Biol. Chem. 2004; 279: 36562-36569Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Schagger H. de Coo R. Bauer M.F. Hofmann S. Godinot C. Brandt U. J. Biol. Chem. 2004; 279: 36349-36353Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Supercomplex architecture suggests a kinetic advantage that increases the respiratory rate by providing substrate channeling between components of the supercomplex as well as stabilizing the complexes (5Schagger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1040) Google Scholar, 14Bianchi C. Genova M.L. Parenti Castelli G. Lenaz G. J. Biol. Chem. 2004; 279: 36562-36569Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Schagger H. de Coo R. Bauer M.F. Hofmann S. Godinot C. Brandt U. J. Biol. Chem. 2004; 279: 36349-36353Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 16Genova M.L. Bianchi C. Lenaz G. Ital. J. Biochem. 2003; 52: 58-61PubMed Google Scholar). There is also compelling evidence to show interdependence among the individual components of supercomplexes. In mammalian cell lines complex III assembly is required to maintain an intact complex I (17Acin-Perez R. Bayona-Bafaluy M.P. Fernandez-Silva P. Moreno-Loshuertos R. Perez-Martos A. Bruno C. Moraes C.T. Enriquez J.A. Mol. Cell. 2004; 13: 805-815Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Structural defects in complex III affected the amount of complex I, whereas chemical inhibition did not. Patients with defects in cytochrome b not only lose complex III, but also show decreased amounts of complex I, while maintaining normal enzymatic activity of the complex (15Schagger H. de Coo R. Bauer M.F. Hofmann S. Godinot C. Brandt U. J. Biol. Chem. 2004; 279: 36349-36353Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Conversely, the disruption of complex I function caused by nonsense mutations in NDUFS4, a subunit of this large multimeric complex, leads to the partial loss of complex III activity in skin fibroblast cultures obtained from Leigh-like patients (18Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (163) Google Scholar, 19Scacco S. Petruzzella V. Budde S. Vergari R. Tamborra R. Panelli D. van den Heuvel L.P. Smeitink J.A. Papa S. J. Biol. Chem. 2003; 278: 44161-44167Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, defects in the complex I subunit ND5 did not cause a loss of complex III in the I-III supercomplex (20Cardol P. Boutaffala L. Memmi S. Devreese B. Matagne R.F. Remacle C. Biochim. Biophys. Acta. 2008; 1777: 388-396Crossref PubMed Scopus (30) Google Scholar). In most eukaryotes cytochrome c oxidase (COX), or complex IV, contains 10 nuclear and 3 mitochondrial encoded subunits. In cell lines, complex IV stabilizes the assembly of complex I. COX10 knock-out mouse cell lines, which were unable to assemble complex IV, showed decreased amounts of complex I, as detected by Western blot analysis following blue native gel electrophoresis. In human cell lines, high COX I mutation levels can lead to destabilization of complex I (11D'Aurelio M. Gajewski C.D. Lenaz G. Manfredi G. Hum. Mol. Genet. 2006; 15: 2157-2169Crossref PubMed Scopus (104) Google Scholar). In addition, inhibition of COX IV expression caused impairment in complex I assembly in mouse cell lines (21Li Y. D'Aurelio M. Deng J.H. Park J.S. Manfredi G. Hu P. Lu J. Bai Y. J. Biol. Chem. 2007; 282: 17557-17562Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Although evidence supports interdependence among MRC complexes, the mechanism regulating this phenomenon remains unclear. We hypothesized that supercomplexes exist in the nematode Caenorhabditis elegans. We also hypothesized that decreasing amounts of complex IV subunits would inhibit the assembly of supercomplexes that include complex IV. We expected that, ultimately, reduced levels of complex IV subunits would in turn reduce amounts of associated components of any supercomplex that includes complex IV. These defects could lead to whole animal phenotypes characteristic of mitochondrial dysfunction. Here we use RNA interference (RNAi) to knock down two different predicted subunits of COX. COX IV and Va are two nuclear-encoded subunits of complex IV. Each controls mitochondrial energy metabolism (22Napiwotzki J. Shinzawa-Itoh K. Yoshikawa S. Kadenbach B. Biol. Chem. 1997; 378: 1013-1021Crossref PubMed Scopus (92) Google Scholar, 23Kadenbach B. Frank V. Rieger T. Napiwotzki J. Mol. Cell. Biochem. 1997; 174: 131-135Crossref PubMed Scopus (18) Google Scholar). COX IV does so through an allosteric mechanism when the extramitochondrial ATP/ADP ratio is high. Binding of ATP to the cytosolic domain of COX IV leads to an increase of the Km of cytochrome c (24Napiwotzki J. Kadenbach B. Biol. Chem. 1998; 379: 335-339Crossref PubMed Scopus (84) Google Scholar), which slows mitochondrial respiration. This mechanism can be abolished by the binding of 3,5-diiodo-thyronine to COX Va (25Arnold S. Goglia F. Kadenbach B. Eur. J. Biochem. 1998; 252: 325-330Crossref PubMed Scopus (172) Google Scholar). No animals with nuclear-encoded genetic defects in subunits of complex IV have been reported. Genes predicted to encode the worm homologues of COX IV and COX Va were each subjected to RNAi knockdown. After knockdown, whole animal phenotypes were recorded, as well as function and structure of the MRC. Lifespan and fecundity of the animals were reduced, consistent with mitochondrial dysfunction. Complex IV activity was reduced, commensurate with decreased amounts of complex IV. Surprisingly, we found that the rate of electron transport within complex I was also decreased by knockdown of either complex IV transcript. Despite the decreased enzymatic activity of complex I in the complex IV knockdown animals, the total amount of complex I remained normal. In diagnosis of patients, our results caution that primary complex IV defects could be misinterpreted to be primary complex I-complex IV defects. Wild type C. elegans (N2) and the Escherichia coli strain HT115 were obtained from the Caenorhabditis Genetics Center. HT115 bacteria containing plasmids used for RNAi knockdown were obtained from GeneService, United Kingdom. Each culture of worms grown for analysis of any sort either had a degree of knockdown measured by qRT-PCR (see below), or lack of complex IV function confirmed by measuring rates of oxygen consumption by mitochondria using TMPD/ascorbate as an electron donor (see below). N2 was grown on either W09C5.8 or Y37D8A.14, the 2 clones of E. coli containing the plasmids that express RNAi corresponding to the target genes, COX IV and COX Va, respectively. The control worms were fed HT115 on Nematode Growth Medium plates (3 days) and transferred to liquid culture (3 additional days) when most animals were young adults. Worm preparation and mitochondrial isolation were performed as previously described (26Kayser E.B. Morgan P.G. Hoppel C.L. Sedensky M.M. J. Biol. Chem. 2001; 276: 20551-20558Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 27Kayser E.B. Sedensky M.M. Morgan P.G. Mech. Ageing Dev. 2004; 125: 455-464Crossref PubMed Scopus (90) Google Scholar). The knockdown worms required 4 and 6 days on the plates and in liquid culture, respectively. In this manner two generations of worms were exposed to RNAi or control bacteria. 5 mm isopropyl β-d-1-thiogalactopyranoside was used to induce RNAi synthesis from the bacteria. Temperature was controlled at 20 °C throughout and the cultures were never allowed to clear. Gene knockdown was checked by qPCR. RNA isolation and quantitative reverse transcriptase-PCR were done by standard techniques. Life Span-Nematodes were grown for two generations on the RNAi strain being studied or the control strain, OP50. Adult nematodes were allowed to lay eggs for 4-6 h on plates containing lawns of the control or RNAi producing bacteria. The adults were removed and the eggs were allowed to hatch. The day of egg laying for this F1 generation was defined as day 0 of life. Animals were moved to new plates on day 3 of life and plated at a density of 20-22 animals per 35-mm plate. The animals were then moved every 2 days. Death, defined as failure to respond to a light touch, was scored each day. Each experiment consisted of 100-150 animals and was repeated in triplicate. The total number of animals for the three experiments was recorded. Growth Rate-In the experiments described for lifespan, the first day of adulthood was recorded by scoring animals for the presence of eggs. Fecundity-Animals were grown on a bacterial lawn of either HT115 or one of the RNAi clones for two generations. Single larvae were then transferred to new plates with the appropriate lawn. These animals were moved every 2 days and their off-spring were counted on the plates from which they were transferred. Offspring from 15 such animals were used to determine average number of offspring. Behavior in Anesthetic-Freshly washed young adult worms from liquid cultures of each strain were transferred to agar plates and exposed to varying concentrations of the volatile anesthetic, halothane to determine the EC50 values (effective concentration at which 50% of the animals are immobilized). Dose-response analysis is performed as previously described (28Morgan P.G. Sedensky M.M. Meneely P.M. Cascorbi H.F. Anesthesiology. 1988; 69: 246-251Crossref PubMed Scopus (37) Google Scholar). Polarography Assay-The polarographic measurement of oxygen consumption of intact mitochondria by a Clark electrode was performed as previously described (29Hoppel C. DiMarco J.P. Tandler B. J. Biol. Chem. 1979; 254: 4164-4170Abstract Full Text PDF PubMed Google Scholar). Malate, succinate, and TMPD/ascorbate were used as the electron donors to complex I-, complex II- and complex IV-dependent respiration, respectively. The complex IV rate was measured in the presence of high ADP and 2,4-dinitrophenol to maximize respiratory capacity of complex IV. Electron Transport Chain (ETC) Assay-Enzyme activities were measured at 30 °C, exclusively using cholate-treated mitochondria (1 mg of mitochondrial protein solubilized in 0.1 m potassium phosphate buffer containing 1% cholate (w/v), pH 7.4, to a final volume of 1 ml). No ETC measurements were made on mitochondrial components prepared for or isolated from blue native gels. CI (NADH-decylubiquinone oxidoreductase (rotenone-sensitive)) (30Hatefi Y. Methods Enzymol. 1978; 53: 11-14Crossref PubMed Scopus (187) Google Scholar), NFR (NADH-ferricyanide oxidoreductase (with background correction)) (30Hatefi Y. Methods Enzymol. 1978; 53: 11-14Crossref PubMed Scopus (187) Google Scholar), CI-III (NADH-cytochrome c oxidoreductase (rotenone-sensitive)) (31Hoppel C. Cooper C. Arch. Biochem. Biophys. 1969; 135: 173-183Crossref PubMed Scopus (32) Google Scholar), CII-III (succinate-cytochrome c oxidoreductase (antimycin-sensitive)) (32Sottocasa G.L. Kuylenstierna B. Ernster L. Bergstrand A. J. Cell Biol. 1967; 32: 415-438Crossref PubMed Scopus (1816) Google Scholar), CIII (Ubiquinol-cytochrome c oxidoreductase (antimycin-sensitive)) (33Krahenbuhl S. Chang M. Brass E.P. Hoppel C.L. J. Biol. Chem. 1991; 266: 20998-21003Abstract Full Text PDF PubMed Google Scholar), and CIV (cytochrome c-oxygen oxidoreductase (KCN-sensitive)) (33Krahenbuhl S. Chang M. Brass E.P. Hoppel C.L. J. Biol. Chem. 1991; 266: 20998-21003Abstract Full Text PDF PubMed Google Scholar) were determined by established spectrophotometric methods as mentioned. The activity of CIV was determined by first-order kinetic and reported as absorbance units/min/mg of protein. The activities of the other assays were determined by zero order kinetic and reported as nanomole of substrate/min/mg of protein. Agilent 8453 UV-visible Spectroscopy System and Agilent ChemStation for UV-visible Spectroscopy (Agilent Technologies Inc, Santa Clara, CA) were the hardware and the software used for the ETC assay, respectively. Blue Native Gel (BNG) Electrophoresis and In-gel Activity Assay (IGA)-BNGs (34Schagger H. Methods Enzymol. 1996; 264: 555-566Crossref PubMed Google Scholar) were loaded with 150-250 μg of mitochondrial protein, determined by Lowry assay, and subjected to membrane solubilization. Digitonin was used with a detergent/protein mass ratio of 6/1. The solubilization was performed at room temperature for 10 min, followed by centrifugation at 15,000 × g at 4 °C for 20 min. After centrifugation, supernatants were collected and Coomassie Blue G-250 was added to the supernatants to obtain a dye/detergent mass ratio of 8/1 before loading onto a 3.5-11% polyacrylamide gradient gel, 0.15 × 14 × 16 cm (Hoefer Inc., Holliston, MA). The gels were run at 100 V until all samples entered the separating gel and then at 300 V for the remainder. When the dye reached one-third of the gel, the first cathode buffer was replaced by the second cathode buffer that contained a 10-fold dilution of Coomassie Blue G-250. Complex I in-gel activity (CI-IGA) was visualized by incubating the gel with 0.5 mm nitro blue tetrazolium and 5 mm NADH in 50 mm Tris-HCl, pH 7.4, at room temperature for 60 min. Complex IV in-gel activity (CIV-IGA) was visualized by incubating the gel with 0.1% (w/v) 3,3′-diaminobenzidine, 0.1% (w/v) cytochrome c and 24 units/ml catalase in 50 mm Tris-HCl, pH 7.4, at 37 °C for 3-6 h. Both protocols are modifications of Grad and Lemire (35Grad L.I. Lemire B.D. Biochim. Biophys. Acta. 2006; 1757: 115-122Crossref PubMed Scopus (45) Google Scholar). To visualize complex I as an individual complex in BNGs, Triton X-100 with a detergent/protein mass ratio of 5/1 was used instead of digitonin. 25 μg of mitochondrial protein were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline/Tween 20 (0.1%) and then incubated with either anti-NDUFS3 (MS112, Mitosciences, Eugene, OR) or anti-adenosine nucleotide transporter (MSA02, Mitosciences). Secondary antibodies were from Santa Cruz Biotechnology. Chemiluminescence substrate (SuperSignal® West Pico, Pierce) was used to develop the reactions. ImageQuant (TL version 2005) software from Amersham Biosciences was used to quantify levels of expression. The expression levels of protein reacting with anti-NDUFS3 were normalized to that of anti-adenosine nucleotide transporter. BNGs were analyzed following the standard mass spectrometry procedure (36Conway J.P. Kinter M. Mol. Cell. Proteomics. 2005; 4: 1522-1540Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In brief, the in-gel tryptic digests of the bands cut from BNGs were analyzed using a capillary column LC-tandem mass spectrometer. An LCQ-Deca ion trap mass spectrometer system (ThermoFinnigan, San Jose, CA) was equipped with a microelectrospray ionization source (Protana, Odense, Denmark), which was operated under microspray conditions at a flow rate of 0.2 μl/min. The digests were analyzed using the data-dependent mode, recording a mass spectrum and three collision-induced dissociation spectra of ions ranging in abundance over several orders or magnitude. The entire sets of collision-induced dissociation spectra in each digest were subjected to data base search against the NCBI data base using the C. elegans taxonomy filter. Analysis of variance was used to analyze groups of data for significant differences. Unpaired Student’s t test was employed to calculate statistical significance of specific pairs if a difference was noted with analysis of variance. Standard deviations (S.D.) and n listed under supplemental data are to enable the reader to determine significance. Error bars in the figures represent mean ± S.E. for each experiment. RNAi-The average percentages of COX IV and COX Va knockdown determined by qPCR from 4 independent cultures were 53.1 ± 7 and 27.4 ± 4%, respectively (see below, and Fig. 1). Phenotypes-Nematodes treated with RNAi for either subunit COX IV (W09C5.8) or COX Va (Y37D8A.14) required an additional day to reach adulthood compared with N2. Lifespan was significantly shortened by 3-5 days, and fecundity was less than half of normal (Table 1). No change in anesthetic sensitivity was seen.TABLE 1Phenotypical study of COX IV and COX Va knockdown wormsPhenotypeN2COX IVCOX VaMean lifespan (days)17 ± 1.114 ± .912 ± 1.5Facundity (eggs/worm)254 ± 22124 ± 14107 ± 17Days to adulthood (days)3 ± 0.14 ± 0.34 ± 0.4EC50 (halothane)3.2 ± 0.22.9 ± 0.22.8 ± 0.2 Open table in a new tab Polarography-MRC function of intact mitochondria isolated from the knockdown worms and N2 was determined by rates of oxygen consumption. As expected, complex IV respiration was decreased by both RNAi knockdowns (Fig. 1). In addition, the state 3 rates of both complex I- and complex II-dependent oxidative phosphorylation were significantly lower in COX IV knockdown worms than in N2. COX Va knockdown significantly decreased complex I-dependent rates but no significant change was seen in the complex II-dependent rates. The defects in MRC function in both RNAi worms were consistent with complex IV deficiencies. The amount of knockdown of the target gene correlated with TMPD/ascorbate rates; respiratory rates were lowest in those preparations with the greatest RNAi effect (Fig. 1B). Electron Transport Chain-KCN-sensitive cytochrome c oxidase activities in COX IV and COX Va knockdown worms were significantly lower than in the wild type, as expected (Fig. 2A). Interestingly, the average rotenone-sensitive NADH-decylubiquinone oxidoreductase activities (electron flow through complex I) in the COX IV and COX Va worms were 59 and 48% lower than in the wild type, respectively (Fig. 2B). NADH-ferricyanide reductase (NFR), a measure of proximal complex I function, was not decreased (Fig. 2C). However, rotenone-sensitive NADH-cytochrome c oxidoreductase (CI-III), a measure of electron flow through complex I to III was decreased in COX Va knockdowns (Fig. 2D). When analyzed by analysis of variance, complex II-III rates in the knockdowns (Fig. 2E, CII-III) were statistically unchanged compared with control. Antimycin A-sensitive decylubiquinol-cytochrome c oxidoreductase activities (complex III) in the knockdown worms were also not statistically different from wild type (Fig. 2F). Because II-III and III activity were unchanged, we concluded that complex II is unaffected by knockdown of complex IV. Complex I and complex IV activity were specifically decreased in the mutants; we therefore asked whether the amounts of these complexes were also changed. Supercomplex Organization in C. elegans Mitochondria-BNGs were used to determine the amount of MRC complexes in the worms with and without RNAi treatment. To optimize the BNG conditions, we first tested dodecyl maltoside (MS), Triton X-100 (TX), and digitonin (DT), as detergents in our isolation (Fig. 3 and supplementary data Fig. S1). We also varied the protein to detergent mass ratio in multiple attempts to optimally isolate supercomplexes as discreet entities. Digitonin at a detergent/protein mass ratio of 6/1 best preserved most MRC supercomplexes (Fig. 3 and supplementary data). Mass spectrometry was used to identify the polypeptides in each band appearing in digitonin (6/1) BNGs. Of the MRC proteins, 26 complex I subunits, 5 complex III subunits, 6 complex IV subunits, and 12 complex V subunits were identified (Table 2). Mitochondrial matrix proteins were found in the same bands as MRC supercomplexes, although the ratio of non-MRC/MRC peptides was less than 1/4, and most commonly seen in isolated complex III. The presence of specific subunits led to the identification of the complexes contained in each band.TABLE 2MRC proteins identified by mass spectrometry in each protein band in digitonin-based BNGs Open table in a new tab In the digitonin gel, multiple bands of greater than 1,236 kDa contained subunits of supercomplex I:III:IV (Fig. 3A, DT) (Table 2). Two other bands at 1,048 and 1,000 kDa were also identified as supercomplex I:III:IV. The 950-kDa band contained only supercomplex I:III. Three bands at 880, 800, and 720 kDa were identified as complex V. Molecular mass analysis predicts that the 880-kDa band represents dimeric complex V, whereas the 720-kDa band is the monomer. Each band contained peptides from all complex V subunits. Relative amounts of these three bands to each other varied slightly in digitonin-based gels, but the sum of their staining was relatively constant (data not shown). The band at 600 kDa corresponds to dimeric complex III. Complex IV is located at 420 kDa, predicted by molecular mass to be dimeric complex IV. A band at 500 kDa, which is positive for CIV-IGA, was not analyzed by mass spectrometry. However, its molecular mass matches the summation of complex III and complex IV (300 and 200 kDa, respectively). Although complex III is usually found only in dimeric form, we cannot rule out that a monomer may form with complex IV. Thus, the band at 500 kDa may represent a supercomplex III:IV but with the monomeric form of each. We also identified bands from the Triton X-100-treated lane (supplementary data). Notably, we found that the band at about 780 kDa contained only complex I subunits, with no proteins identified from other complexes or from non-MRC proteins. Thus, Triton X-100 can be used to isolate complex I, although it is not known whether it contains a complete form of the complex. The two bands at ∼875-900 kDa each contained complex I and III subunits. No bands were seen above 900 kDa in both Triton X-100 and dodecyl maltoside lanes. We were not able to detect complex II in any of our BNGs. We used in-gel activity assays of BNGs of N2 mitochondria to verify the presence and amount of MRC complexes. IGAs (Fig. 3, B and C) corroborated the proteomic results. No complex I or complex IV activity was seen outside of bands that were predicted by mass spectrometry to contain subunits of these complexes. Complex I remained sufficiently intact in all three detergents to maintain enzymatic function, whereas complex IV activity was only seen in the digitonin-treated lane. COX IV and COX Va Knockdowns-RNAi treatment for COX IV and COX Va disrupted supercomplex formation (Fig. 4), as seen in BNGs of digitonin-treated mitochondria. Not surprisingly, COX IV and COX Va knockdown decreased amounts of dimeric complex IV (Fig. 4A). Supercomplex I:III:IV was also decreased in both knockdowns, compared with the wild type, whereas supercomplex I:III was increased. RNAi treatment did not affect amounts of isolated complex III or complex V. CIV-IGA verified loss of complex IV in the knockdown animals (Fig. 4B). Compared with wild type activity, we observed a decrease in COX activity at 420 kDa, the dimeric complex IV, in both RNAi worm strains. The activity of the complex IV component in supercomplex I:II-I:IV was also decreased in the RNAi-treated worms. CI-IGA was decreased in supercomplex I:III:IV after RNAi treatment in the knockdown worms, consistent with the decrease in supercomplex I:III:IV formation (Fig. 4C). However, CI-IGA of supercomplex I:III increased. The ratio of CI-IGA activity in I:III:IV to the total CI-IGA, a" @default.
• W2076055995 created "2016-06-24" @default.
• W2076055995 creator A5000879558 @default.
• W2076055995 creator A5006598602 @default.
• W2076055995 creator A5011148894 @default.
• W2076055995 creator A5043953340 @default.
• W2076055995 date "2009-03-01" @default.
• W2076055995 modified "2023-09-30" @default.
• W2076055995 title "Complex I Function Is Defective in Complex IV-deficient Caenorhabditis elegans" @default.
• W2076055995 cites W1191980701 @default.
• W2076055995 cites W1486967557 @default.
• W2076055995 cites W1532909424 @default.
• W2076055995 cites W1537763986 @default.
• W2076055995 cites W1541314725 @default.
• W2076055995 cites W1673990805 @default.
• W2076055995 cites W1966728800 @default.
• W2076055995 cites W1969805452 @default.
• W2076055995 cites W1976436058 @default.
• W2076055995 cites W1982753455 @default.
• W2076055995 cites W1995042253 @default.
• W2076055995 cites W1999749322 @default.
• W2076055995 cites W2000513681 @default.
• W2076055995 cites W2001799565 @default.
• W2076055995 cites W2005045122 @default.
• W2076055995 cites W2005113567 @default.
• W2076055995 cites W2005280808 @default.
• W2076055995 cites W2006274062 @default.
• W2076055995 cites W2013858150 @default.
• W2076055995 cites W2019169693 @default.
• W2076055995 cites W2028860264 @default.
• W2076055995 cites W2030760120 @default.
• W2076055995 cites W2031972450 @default.
• W2076055995 cites W2035665637 @default.
• W2076055995 cites W2036161666 @default.
• W2076055995 cites W2043947564 @default.
• W2076055995 cites W2044747701 @default.
• W2076055995 cites W2056932327 @default.
• W2076055995 cites W2064847009 @default.
• W2076055995 cites W2064907146 @default.
• W2076055995 cites W2065683829 @default.
• W2076055995 cites W2073947021 @default.
• W2076055995 cites W2091339923 @default.
• W2076055995 cites W2092871687 @default.
• W2076055995 cites W2094844362 @default.
• W2076055995 cites W2096254067 @default.
• W2076055995 cites W2097156878 @default.
• W2076055995 cites W2111873608 @default.
• W2076055995 cites W2131226021 @default.
• W2076055995 cites W2136895391 @default.
• W2076055995 cites W2137134977 @default.
• W2076055995 cites W2138115215 @default.
• W2076055995 cites W2152284208 @default.
• W2076055995 cites W2155265427 @default.
• W2076055995 cites W2159139985 @default.
• W2076055995 cites W2161347267 @default.
• W2076055995 cites W2163800311 @default.
• W2076055995 cites W2171628789 @default.
• W2076055995 cites W4253146696 @default.
• W2076055995 doi "https://doi.org/10.1074/jbc.m805733200" @default.
• W2076055995 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2649102" @default.
• W2076055995 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19074434" @default.
• W2076055995 hasPublicationYear "2009" @default.
• W2076055995 type Work @default.
• W2076055995 sameAs 2076055995 @default.
• W2076055995 citedByCount "71" @default.
• W2076055995 countsByYear W20760559952012 @default.
• W2076055995 countsByYear W20760559952013 @default.
• W2076055995 countsByYear W20760559952014 @default.
• W2076055995 countsByYear W20760559952015 @default.
• W2076055995 countsByYear W20760559952016 @default.
• W2076055995 countsByYear W20760559952017 @default.
• W2076055995 countsByYear W20760559952018 @default.
• W2076055995 countsByYear W20760559952019 @default.
• W2076055995 countsByYear W20760559952020 @default.
• W2076055995 countsByYear W20760559952021 @default.
• W2076055995 countsByYear W20760559952022 @default.
• W2076055995 countsByYear W20760559952023 @default.
• W2076055995 crossrefType "journal-article" @default.
• W2076055995 hasAuthorship W2076055995A5000879558 @default.
• W2076055995 hasAuthorship W2076055995A5006598602 @default.
• W2076055995 hasAuthorship W2076055995A5011148894 @default.
• W2076055995 hasAuthorship W2076055995A5043953340 @default.
• W2076055995 hasBestOaLocation W20760559951 @default.
• W2076055995 hasConcept C104317684 @default.
• W2076055995 hasConcept C14036430 @default.
• W2076055995 hasConcept C185592680 @default.
• W2076055995 hasConcept C2776496032 @default.
• W2076055995 hasConcept C2778944004 @default.
• W2076055995 hasConcept C54355233 @default.
• W2076055995 hasConcept C86803240 @default.
• W2076055995 hasConcept C95444343 @default.
• W2076055995 hasConceptScore W2076055995C104317684 @default.
• W2076055995 hasConceptScore W2076055995C14036430 @default.
• W2076055995 hasConceptScore W2076055995C185592680 @default.
• W2076055995 hasConceptScore W2076055995C2776496032 @default.
• W2076055995 hasConceptScore W2076055995C2778944004 @default.
• W2076055995 hasConceptScore W2076055995C54355233 @default.
• W2076055995 hasConceptScore W2076055995C86803240 @default.

• next