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• W2725547456 abstract "•Absence of TTC19 causes cIII defect and neurological impairment in humans and mice•TTC19 binds fully assembled cIII, after the incorporation of UQCRFS1•UQCRFS1 undergoes proteolytic processing after its incorporation in cIII•TTC19 is involved in the clearance of UQCRFS1 fragments that inhibit cIII Loss-of-function mutations in TTC19 (tetra-tricopeptide repeat domain 19) have been associated with severe neurological phenotypes and mitochondrial respiratory chain complex III deficiency. We previously demonstrated the mitochondrial localization of TTC19 and its link with complex III biogenesis. Here we provide detailed insight into the mechanistic role of TTC19, by investigating a Ttc19−/− mouse model that shows progressive neurological and metabolic decline, decreased complex III activity, and increased production of reactive oxygen species. By using both the Ttc19−/− mouse model and a range of human cell lines, we demonstrate that TTC19 binds to the fully assembled complex III dimer, i.e., after the incorporation of the iron-sulfur Rieske protein (UQCRFS1). The in situ maturation of UQCRFS1 produces N-terminal polypeptides, which remain bound to holocomplex III. We show that, in normal conditions, these UQCRFS1 fragments are rapidly removed, but when TTC19 is absent they accumulate within complex III, causing its structural and functional impairment. Loss-of-function mutations in TTC19 (tetra-tricopeptide repeat domain 19) have been associated with severe neurological phenotypes and mitochondrial respiratory chain complex III deficiency. We previously demonstrated the mitochondrial localization of TTC19 and its link with complex III biogenesis. Here we provide detailed insight into the mechanistic role of TTC19, by investigating a Ttc19−/− mouse model that shows progressive neurological and metabolic decline, decreased complex III activity, and increased production of reactive oxygen species. By using both the Ttc19−/− mouse model and a range of human cell lines, we demonstrate that TTC19 binds to the fully assembled complex III dimer, i.e., after the incorporation of the iron-sulfur Rieske protein (UQCRFS1). The in situ maturation of UQCRFS1 produces N-terminal polypeptides, which remain bound to holocomplex III. We show that, in normal conditions, these UQCRFS1 fragments are rapidly removed, but when TTC19 is absent they accumulate within complex III, causing its structural and functional impairment. Complex III, or ubiquinol:cytochrome c oxidoreductase, is the central complex in the mitochondrial electron transport chain. Complex III is a multi-heteromeric enzyme, organized in a symmetrical dimeric structure (cIII2) of ∼480 kDa (Iwata et al., 1998Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex.Science. 1998; 281: 64-71Crossref PubMed Scopus (1062) Google Scholar). cIII2 catalyzes the transfer of electrons from Coenzyme Q to cytochrome c, while pumping protons from the matrix to the intermembrane space. In yeast and birds, the cIII monomer is composed of ten different subunits, whereas an eleventh subunit, Subunit 9 (Su9) in the bovine nomenclature, has been identified in mammals as a post-translational proteolytic product consisting of the 78-amino acid-long N-terminal mitochondrial targeting sequence (MTS) of the 2Fe-2S cluster-containing Rieske protein, encoded by the UQCRFS1 gene (Brandt et al., 1993Brandt U. Yu L. Yu C.A. Trumpower B.L. The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.J. Biol. Chem. 1993; 268: 8387-8390Abstract Full Text PDF PubMed Google Scholar). Cytochrome b (MT-CYB), the only cIII2 component encoded by mtDNA, is one of the three catalytic subunits of the complex, and it contains a low-potential (bL) and a high-potential (bH) heme b moieties as prosthetic groups. Of the other ten subunits, encoded by nine nuclear genes, two play a catalytic role: the UQCRFS1 Rieske protein and cytochrome c1, encoded by CYC1, which contains a c-type heme group. The role of the other eight supernumerary subunits remains unclear. Although cIII2 assembly has been less intensively investigated compared to other respiratory complexes, in the last few years the study of yeast mutant models has provided some insight into this process (Gruschke et al., 2012Gruschke S. Römpler K. Hildenbeutel M. Kehrein K. Kühl I. Bonnefoy N. Ott M. The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria.J. Cell Biol. 2012; 199: 137-150Crossref PubMed Scopus (52) Google Scholar, Hildenbeutel et al., 2014Hildenbeutel M. Hegg E.L. Stephan K. Gruschke S. Meunier B. Ott M. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation.J. Cell Biol. 2014; 205: 511-524Crossref PubMed Scopus (50) Google Scholar, Smith et al., 2012Smith P.M. Fox J.L. Winge D.R. Biogenesis of the cytochrome bc(1) complex and role of assembly factors.Biochim. Biophys. Acta. 2012; 1817: 276-286Crossref PubMed Scopus (83) Google Scholar, Zara et al., 2009Zara V. Conte L. Trumpower B.L. Biogenesis of the yeast cytochrome bc1 complex.Biochim. Biophys. Acta. 2009; 1793: 89-96Crossref PubMed Scopus (72) Google Scholar). Studies on cIII2-associated human diseases have confirmed that some cIII2 assembly steps are similar to yeast, especially the late ones. Based on the yeast model, cIII2 assembly starts with the insertion into the inner mitochondrial membrane of MT-CYB, bound to chaperones UQCC1, UQCC2, and UQCC3. These chaperones are released during the sequential incorporation of additional subunits into an inactive dimeric pre-cIII2, which is eventually activated by the incorporation of the last subunits, UQCRFS1 and UQCR11. This final step, i.e., the UQCRFS1 incorporation, is mediated by BCS1L, the most extensively characterized cIII2 assembly factor, and LYRM7 (MZM1L), a matrix protein that stabilizes the subunit before its assembly into cIII2 (Fernández-Vizarra and Zeviani, 2015Fernández-Vizarra E. Zeviani M. Nuclear gene mutations as the cause of mitochondrial complex III deficiency.Front. Genet. 2015; 6: 134Crossref PubMed Scopus (95) Google Scholar). Pathogenic mutations have been found in some of cIII2-related ancillary factors, including BCS1L (de Lonlay et al., 2001de Lonlay P. Valnot I. Barrientos A. Gorbatyuk M. Tzagoloff A. Taanman J.W. Benayoun E. Chrétien D. Kadhom N. Lombès A. et al.A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy, and liver failure.Nat. Genet. 2001; 29: 57-60Crossref PubMed Scopus (263) Google Scholar, Fellman, 2002Fellman V. The GRACILE syndrome, a neonatal lethal metabolic disorder with iron overload.Blood Cells Mol. Dis. 2002; 29: 444-450Crossref PubMed Scopus (54) Google Scholar, Fernandez-Vizarra et al., 2007Fernandez-Vizarra E. Bugiani M. Goffrini P. Carrara F. Farina L. Procopio E. Donati A. Uziel G. Ferrero I. Zeviani M. Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy.Hum. Mol. Genet. 2007; 16: 1241-1252Crossref PubMed Scopus (133) Google Scholar, Hinson et al., 2007Hinson J.T. Fantin V.R. Schönberger J. Breivik N. Siem G. McDonough B. Sharma P. Keogh I. Godinho R. Santos F. et al.Missense mutations in the BCS1L gene as a cause of the Björnstad syndrome.N. Engl. J. Med. 2007; 356: 809-819Crossref PubMed Scopus (160) Google Scholar, Morán et al., 2010Morán M. Marín-Buera L. Gil-Borlado M.C. Rivera H. Blázquez A. Seneca S. Vázquez-López M. Arenas J. Martín M.A. Ugalde C. Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.Hum. Mutat. 2010; 31: 930-941Crossref PubMed Scopus (52) Google Scholar, Ramos-Arroyo et al., 2009Ramos-Arroyo M.A. Hualde J. Ayechu A. De Meirleir L. Seneca S. Nadal N. Briones P. Clinical and biochemical spectrum of mitochondrial complex III deficiency caused by mutations in the BCS1L gene.Clin. Genet. 2009; 75: 585-587Crossref PubMed Scopus (31) Google Scholar), TTC19 (Ardissone et al., 2015Ardissone A. Granata T. Legati A. Diodato D. Melchionda L. Lamantea E. Garavaglia B. Ghezzi D. Moroni I. Mitochondrial complex III deficiency caused by TTC19 defects: report of a novel mutation and review of literature.JIMD Rep. 2015; 22: 115-120Crossref PubMed Scopus (13) Google Scholar, Atwal, 2014Atwal P.S. Mutations in the complex III assembly factor tetratricopeptide 19 gene TTC19 are a rare cause of Leigh syndrome.JIMD Rep. 2014; 14: 43-45Crossref PubMed Scopus (23) Google Scholar, Ghezzi et al., 2011Ghezzi D. Arzuffi P. Zordan M. Da Re C. Lamperti C. Benna C. D’Adamo P. Diodato D. Costa R. Mariotti C. et al.Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies.Nat. Genet. 2011; 43: 259-263Crossref PubMed Scopus (128) Google Scholar, Kunii et al., 2015Kunii M. Doi H. Higashiyama Y. Kugimoto C. Ueda N. Hirata J. Tomita-Katsumoto A. Kashikura-Kojima M. Kubota S. Taniguchi M. et al.A Japanese case of cerebellar ataxia, spastic paraparesis, and deep sensory impairment associated with a novel homozygous TTC19 mutation.J. Hum. Genet. 2015; 60: 187-191Crossref PubMed Scopus (12) Google Scholar, Melchionda et al., 2014Melchionda L. Damseh N.S. Abu Libdeh B.Y. Nasca A. Elpeleg O. Zanolini A. Ghezzi D. A novel mutation in TTC19 associated with isolated complex III deficiency, cerebellar hypoplasia, and bilateral basal ganglia lesions.Front. Genet. 2014; 5: 397Crossref PubMed Scopus (13) Google Scholar, Mordaunt et al., 2015Mordaunt D.A. Jolley A. Balasubramaniam S. Thorburn D.R. Mountford H.S. Compton A.G. Nicholl J. Manton N. Clark D. Bratkovic D. et al.Phenotypic variation of TTC19-deficient mitochondrial complex III deficiency: a case report and literature review.Am. J. Med. Genet. A. 2015; 167: 1330-1336Crossref PubMed Scopus (26) Google Scholar, Morino et al., 2014Morino H. Miyamoto R. Ohnishi S. Maruyama H. Kawakami H. Exome sequencing reveals a novel TTC19 mutation in an autosomal recessive spinocerebellar ataxia patient.BMC Neurol. 2014; 14: 5Crossref PubMed Scopus (27) Google Scholar, Nogueira et al., 2013Nogueira C. Barros J. Sá M.J. Azevedo L. Taipa R. Torraco A. Meschini M.C. Verrigni D. Nesti C. Rizza T. et al.Novel TTC19 mutation in a family with severe psychiatric manifestations and complex III deficiency.Neurogenetics. 2013; 14: 153-160Crossref PubMed Scopus (34) Google Scholar), LYRM7 (Dallabona et al., 2016Dallabona C. Abbink T.E. Carrozzo R. Torraco A. Legati A. van Berkel C.G. Niceta M. Langella T. Verrigni D. Rizza T. et al.LYRM7 mutations cause a multifocal cavitating leukoencephalopathy with distinct MRI appearance.Brain. 2016; 139: 782-794Crossref PubMed Scopus (43) Google Scholar, Invernizzi et al., 2013Invernizzi F. Tigano M. Dallabona C. Donnini C. Ferrero I. Cremonte M. Ghezzi D. Lamperti C. Zeviani M. A homozygous mutation in LYRM7/MZM1L associated with early onset encephalopathy, lactic acidosis, and severe reduction of mitochondrial complex III activity.Hum. Mutat. 2013; 34: 1619-1622Crossref PubMed Scopus (50) Google Scholar), UQCC2 (Tucker et al., 2013Tucker E.J. Wanschers B.F. Szklarczyk R. Mountford H.S. Wijeyeratne X.W. van den Brand M.A. Leenders A.M. Rodenburg R.J. Reljić B. Compton A.G. et al.Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.PLoS Genet. 2013; 9: e1004034Crossref PubMed Scopus (77) Google Scholar), and UQCC3 (Wanschers et al., 2014Wanschers B.F. Szklarczyk R. van den Brand M.A. Jonckheere A. Suijskens J. Smeets R. Rodenburg R.J. Stephan K. Helland I.B. Elkamil A. et al.A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity, and cytochrome b stability.Hum. Mol. Genet. 2014; 23: 6356-6365Crossref PubMed Scopus (54) Google Scholar). In particular, mutations in TTC19 have been identified in patients with heterogeneous, but invariably severe, phenotypes, including early-onset, slowly progressive encephalomyopathy; adult-onset, rapidly progressive multisystem neurological failure; adult-onset spinocerebellar ataxia; and childhood or juvenile spinocerebellar ataxia with psychosis (OMIM: 613814). TTC19 encodes the precursor of the tetratricopeptide repeat domain 19 protein. TTC19, a 380-amino acid-long polypeptide, is addressed to mitochondria by a 70-amino acid-long MTS, which is removed after translocation of the protein into the inner mitochondrial compartment. Mature human TTC19 is a 35-kDa protein embedded within the inner mitochondrial membrane, with orthologs in multicellular animals but neither in fungi nor in plants. Unlike other assembly factors, TTC19 binds to mature dimeric cIII2, whereas, in mutant human fibroblasts, its absence leads to the accumulation of lower molecular weight species-containing cIII2 subunits, UQCRC1 and UQCRC2 (Ghezzi et al., 2011Ghezzi D. Arzuffi P. Zordan M. Da Re C. Lamperti C. Benna C. D’Adamo P. Diodato D. Costa R. Mariotti C. et al.Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies.Nat. Genet. 2011; 43: 259-263Crossref PubMed Scopus (128) Google Scholar). Here we present a study that elucidates the molecular role of TTC19 by investigating human cell and mouse animal models. We created a constitutive Ttc19−/− mouse by gene targeting (Figure S1). The gender and genotype distribution at birth was compatible with a mendelian autosomal recessive trait and no evidence of embryonic lethality. At 6 months of age, both Ttc19−/− males and females showed a reduction in body weight compared to wild-type (WT) littermates (Figure 1A). No differences were observed between Ttc19+/+ and Ttc19+/− animals for any of the parameters investigated; therefore, both genotypes were considered as controls (Ttc19WT). Several tests were used to assess neurological, behavioral, and metabolic features. At 6 months of age, Ttc19−/− mice showed a pathological feet-clasping reflex (Figure 1B), and they scored significantly less than WT littermates in rotarod, cylinder, negative geotaxis, and pole tests, which measure motor coordination, exploratory behavior, general proprioception, and motor planning skills, respectively (Figures 1C–1F). Motor endurance, assessed by standard treadmill test, was also reduced in Ttc19−/− versus Ttc19WT animals (Figure 1G). Some of these features were already present at 3 months of age (Figure S2). No differences in lifespan were observed up to 21 months (data not shown). To study whole-body metabolism, we used a comprehensive lab animals monitoring system (CLAMS). Ttc19−/− females showed reduced food and water intake compared to Ttc19WT female littermates (Figures S3A and S3B); VO2 consumption and VCO2 production were significantly reduced in Ttc19−/− versus Ttc19WT animals of both sexes (Figures S3C and S3D); accordingly, energy expenditure, measured as the ratio between heat rate and body weight, was decreased (Figure S3E), although statistical significance was achieved only in females. Likewise, the respiratory exchange ratio (RER) was significantly reduced in females, suggesting increased utilization of fat versus carbohydrates to produce energy (Figure S3F). Total, ambulatory, and rearing movements were significantly decreased in Ttc19−/− versus Ttc19WT littermates of both genders (Figure S3G). These data indicate neurological impairment and reduction in energy metabolism of Ttc19−/− mice. Extensive astrogliosis, most often surrounding dilated vascular structures (Figures S4A and S4C), and the accumulation of ubiquitinated proteins in neurons (Figure S4E) were detected in the thalamus of all Ttc19−/− mice analyzed (n = 6), suggesting an ongoing brain injury. These features were not observed in Ttc19WT littermates (n = 6) (Figures S4B, S4D, and S4F). No abnormalities were detected in skeletal muscle by H&E (data not shown). Light microscopy of brain sections stained by H&E (data not shown) or Nissl (Figure S4G) did not reveal obvious neuronal loss, compared to Ttc19WT brain (Figure S4H). No sign of neurodegeneration or apoptosis was detected by staining brain sections with fluorojade C or terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) (data not shown). Similar to human patients, Ttc19 ablation in mice caused cIII2 deficiency in all tested tissues. At 3 and 6 months of age, cIII2/citrate synthase (CS) activity was significantly reduced (≈50%, p < 0.005) in brain, liver, and skeletal muscle from Ttc19−/− versus Ttc19WT (Figure 2A), with no gender differences. The residual activity was further reduced (≈40%) at 18 months in both females and males (data not shown). Defective cIII2 activity is associated with increased production of reactive oxygen species (ROS) (Diaz et al., 2012Diaz F. Enríquez J.A. Moraes C.T. Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV.Mol. Cell. Biol. 2012; 32: 415-429Crossref PubMed Scopus (96) Google Scholar, Hinson et al., 2007Hinson J.T. Fantin V.R. Schönberger J. Breivik N. Siem G. McDonough B. Sharma P. Keogh I. Godinho R. Santos F. et al.Missense mutations in the BCS1L gene as a cause of the Björnstad syndrome.N. Engl. J. Med. 2007; 356: 809-819Crossref PubMed Scopus (160) Google Scholar), through reverse electron transfer from reduced heme bL (Borek et al., 2008Borek A. Sarewicz M. Osyczka A. Movement of the iron-sulfur head domain of cytochrome bc(1) transiently opens the catalytic Q(o) site for reaction with oxygen.Biochemistry. 2008; 47: 12365-12370Crossref PubMed Scopus (49) Google Scholar, Dröse and Brandt, 2008Dröse S. Brandt U. The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex.J. Biol. Chem. 2008; 283: 21649-21654Crossref PubMed Scopus (281) Google Scholar). Accordingly, H2O2 production in isolated mitochondria from liver, heart, and skeletal muscle was significantly higher in Ttc19−/− compared to Ttc19WT littermates (Figures 2B–2D). Next, the assembly of cIII2 in mitochondria isolated from mouse brain, liver, and skeletal muscle was investigated by blue native gel electrophoresis (BNGE). First dimension (1D)-BNGE revealed an altered electrophoretic pattern in the Ttc19−/− samples from all three tissues (Figure 3A), as the cIII2 band migrated more slowly and appeared blurred. The aberrant band was observed when using either digitonin or dodecyl-maltoside (DDM) as solubilizing detergents (Figure 3B). To further investigate this phenomenon, we carried out complexome profiling of Ttc19−/− versus Ttc19WT isolated liver (data not shown) and brain mitochondria solubilized with 1% DDM (Figure 3C). The 1D-BNGE lanes were excised in 50 slices, and each slice was analyzed by mass spectrometry. In Ttc19−/− mitochondria, cIII2 subunits were detected in an area of the gel slightly wider and with higher apparent molecular mass than cIII2 subunits from Ttc19WT mitochondria. Interestingly, a normal migration pattern was restored by adeno-associated virus serotype 2/8 (AAV2/8)-mediated expression of human 6xHis-tagged wild-type TTC19 (hTTC19His6) in Ttc19−/− mouse liver mitochondria (Figure S5A), demonstrating that the electrophoretic aberrations were specifically due to the absence of Ttc19. No accumulation of cIII2 sub-assemblies was detected in mitochondria from Ttc19−/− mouse tissues by either complexome profiling (Figure 3C) or denaturing second dimension (2D)-BNGE using antibodies against several subunits of cIII2 (Figure S5B). Proteomic analysis of SDS-PAGE of the native BNGE band corresponding to cIII2 or the immunocaptured cIII2 from Ttc19WT in comparison with Ttc19−/− failed to show any difference in protein composition between the two genotypes. However, when the band corresponding to holocomplex cIII2 was isolated and run through a denaturing SDS-PAGE and analyzed by western blot immunodetection, we observed a reduction of incorporated intact UQCRFS1 in Ttc19−/− versus Ttc19WT mouse samples and the accumulation of UQCRFS1 degradation products (Figure 3D). Interestingly, the levels of intact UQCRFS1 incorporated into cIII2 were restored in Ttc19−/− liver (Figure 3E) by expressing hTTC19His6 with the AAV2/8-hTTC19His6 vector. These results demonstrated a role for TTC19 in stabilizing cIII2, through a specific protective effect on UQCRFS1. Next, we investigated the dynamics of incorporation of an early-assembled cIII2 subunit, UQCRB, and of UQCRFS1. The 35S-labeled translation products were incubated with Ttc19−/− and Ttc19WT mouse liver mitochondria at different times (15-, 30-, and 60-min pulses); samples were then washed and incubated for a further 2 hr (chase). The incorporation into cIII2 of both UQCRB and UQCRFS1 was not significantly different between the Ttc19−/− and Ttc19WT samples (Figure S6). We then cut the cIII2 band from the BNGE gel and denatured and electrophoresed it by SDS-PAGE. Radiolabeled UQCRFS1-derived fragments, including 12, 8, and 4 kDa in size, were already evident after a 15-min pulse in both Ttc19−/− and Ttc19WT mitochondria (Figures 4A and 4B ). Densitometric analysis of intact UQCRFS1 versus fragmented UQCRFS1 showed that fragmented UQCRFS1 persisted longer in Ttc19−/− compared to Ttc19WT samples (Figures 4C and 4D). These results indicate that UQCRFS1 is physiologically proteolysed once it is incorporated in cIII2, since fragmented species were bound to cIII2 and present very early in both Ttc19−/− and Ttc19WT samples, but their clearance was slowed down in Ttc19−/− versus Ttc19WT mitochondria (Figures 4C and 4D). The percentage of mature, intact UQCRFS1 was lower in Ttc19−/− versus Ttc19WT at 60 and 180 min, although the difference was not statistically significant (p = 0.067) (Figure 4C). We also analyzed the relative amounts of the UQCRFS1 fragments at different time points. The amount of the 12- and 8-kDa fragments progressively decreased over time, whereas the 4-kDa fragment was virtually unchanged for all the time points of the experiment. However, the decrease in the amount of both 12- and 8-kDa fragments was slower in the Ttc19−/− versus Ttc19WT samples (Figure 4C), and this difference was significant for the 12-kDa fragment at 180 min (p = 0.02) (Figure 4D). The reduced clearance of UQCRFS1 fragments over time can explain their accumulation in steady-state conditions (Figure 3D), and it suggests a specific role for TTC19 as a husbandry factor in the turnover of damaged UQCRFS1 within cIII2. Finally, we observed reduced incorporation of UQCRFS1 in the presence of a general protease inhibitor cocktail, indicating that proteolytic processing is necessary for the correct insertion of this subunit into cIII2. The action of proteases is also necessary for the clearance of the UQCRFS1 fragments, because their relative amounts versus mature UQCRFS1 remained the same as in the 15-min pulse (Figure 4B). To gain insight into the identity of the accumulated fragments derived from UQCRFS1, we again electrophoresed the cIII2 band from three Ttc19WT and three Ttc19−/− samples from liver mitochondria. Each of the gel lanes were cut into slices and analyzed by mass spectrometry after tryptic digestion. The gel was divided into two sections, one encompassing the protein species from 30 to 14 kDa, where the mature UQCRFS1 is found (calculated molecular weight [MW]: 21.5 kDa), whereas the second one encompassed the interval from 14 kDa to the Coomassie blue dye front of the gel, where the UQCRFS1 fragments were detected. In the 30- to 14-kDa section, we consistently detected tryptic peptides covering the full length of processed, mature UQCRFS1, without the 78 N-terminal amino acid-long MTS (Brandt et al., 1993Brandt U. Yu L. Yu C.A. Trumpower B.L. The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.J. Biol. Chem. 1993; 268: 8387-8390Abstract Full Text PDF PubMed Google Scholar). Conversely, in the 14 kDa-dye front section of the gel, peptides corresponding to the MTS/Su9 were consistently detected in both Ttc19WT and Ttc19−/− samples, along with other peptides corresponding to the rest of the UQCRFS1 sequence, with the exception of the C-terminal end (Figure 4D). To further characterize the proposed direct interaction of TTC19 with cIII2 (Ghezzi et al., 2011Ghezzi D. Arzuffi P. Zordan M. Da Re C. Lamperti C. Benna C. D’Adamo P. Diodato D. Costa R. Mariotti C. et al.Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies.Nat. Genet. 2011; 43: 259-263Crossref PubMed Scopus (128) Google Scholar), we performed in organello import experiments using in vitro translated 35S-labeled hTTC19 followed by BNGE. In digitonin-treated samples, we detected 35S-hTTC19 in bands corresponding to the cIII2 holocomplex and cIII2-containing supercomplexes (SCs). Newly incorporated 35S-hTTC19 was detected only in Ttc19−/−, but not in Ttc19WT, mitochondria, suggesting that the interaction of the endogenous Ttc19 with cIII2 prevents its dislodgment by 35S-hTTC19. However, this interaction must be relatively labile, since no 35S-hTTC19 signal was detected in mitochondria treated with DDM, a detergent stronger than digitonin (Figures 5A and 5B ). To test whether the TTC19 migration in BNGE is due to its physical interaction with cIII2, we then performed co-immunoprecipitation assays using a HEK293T cell line expressing a FLAG-tagged recombinant human TTC19 (hTTC19FLAG). We first showed that hTTC19FLAG was robustly expressed in these cells upon doxycycline induction (Figure S7A) and was co-immunoprecipitated with UQCRFS1 and UQCRC2 (Figure S7B). The protein interactions of hTTC19FLAG were further analyzed by quantitative mass spectrometry analysis of mitoplasts from naive and recombinant HEK293T cells after FLAG immunopurification. Comparisons were enabled by stable isotopic labeling of amino acids in cell culture (SILAC). From duplicate, reciprocal-labeling experiments (see the STAR Methods for details), a total of 64 proteins was quantitatively different in the hTTC19FLAG versus naive samples (Figure 5C; Table S1). Of these 64 proteins, 51 were components of the respiratory chain, including eight subunits of cIII2, as well as numerous subunits of cI and cIV (Table S1). These data demonstrate that hTTC19FLAG co-purifies with cIII2 holocomplex, as well as with cIII2-containing SCs. Interestingly, the mitochondrial proteases YME1L and PARL, together with the scaffold protein STOML2 (SLP2), all three members of the SPY complex (Wai et al., 2016Wai T. Saita S. Nolte H. Müller S. König T. Richter-Dennerlein R. Sprenger H.G. Madrenas J. Mühlmeister M. Brandt U. et al.The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.EMBO Rep. 2016; 17: 1844-1856Crossref PubMed Scopus (101) Google Scholar), were also co-immunopurified with TTC19, indicating physical interaction (Table S1). To explore the role of TTC19 in cIII2 biogenesis, we analyzed three different cIII2-deficient cell lines: a TTC19-less human fibroblast cell line (Ghezzi et al., 2011Ghezzi D. Arzuffi P. Zordan M. Da Re C. Lamperti C. Benna C. D’Adamo P. Diodato D. Costa R. Mariotti C. et al.Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies.Nat. Genet. 2011; 43: 259-263Crossref PubMed Scopus (128) Google Scholar), two BCS1L-mutated human fibroblast cell lines (Fernandez-Vizarra et al., 2007Fernandez-Vizarra E. Bugiani M. Goffrini P. Carrara F. Farina L. Procopio E. Donati A. Uziel G. Ferrero I. Zeviani M. Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy.Hum. Mol. Genet. 2007; 16: 1241-1252Crossref PubMed Scopus (133) Google Scholar), and a recombinant HeLa cell line overexpressing MZM1L/LYRM7HA (Sánchez et al., 2013Sánchez E. Lobo T. Fox J.L. Zeviani M. Winge D.R. Fernández-Vizarra E. LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial complex III assembly in human cells.Biochim. Biophys. Acta. 2013; 1827: 285-293Crossref PubMed Scopus (64) Google Scholar). The latter two cell lines showed a strong reduction in the levels of UQCRFS1 present in cIII2 (Figure 6A), since BCS1L is a late-stage assembly factor necessary for UQCRFS1 incorporation (Fernandez-Vizarra et al., 2007Fernandez-Vizarra E. Bugiani M. Goffrini P. Carrara F. Farina L. Procopio E. Donati A. Uziel G. Ferrero I. Zeviani M. Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy.Hum. Mol. Genet. 2007; 16: 1241-1252Crossref PubMed Scopus (133) Google Scholar), whereas overexpression of LYRM7, which physically interacts with UQCRFS1, causes UQCRFS1 to be sequestered in the mitochondrial matrix, thus preventing its incorporation into cIII2 (Sánchez et al., 2013Sánchez E. Lobo T. Fox J.L. Zeviani M. Winge D.R. Fernández-Vizarra E. LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial complex III assembly in human cells.Biochim. Biophys. Acta. 2013; 1827: 285-293Crossref PubMed Scopus (64) Google Scholar). Immunodetection of 1D-BNGE (Figure 6A) and 2D-BNGE (Figure 6B) revealed that the lack of TTC19 did not prevent UQCRFS1 from being incorporated into cIII2 and SC, since comparable levels were found in control and TTC19-less fibroblasts. In addition, a substantial amount of TTC19 co-migrated with cIII2, cIII2 + cIV, and SC in control cell lines (Figure 6B), confirming the data fro" @default.
• W2725547456 created "2017-07-14" @default.
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• W2725547456 title "TTC19 Plays a Husbandry Role on UQCRFS1 Turnover in the Biogenesis of Mitochondrial Respiratory Complex III" @default.
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• W2725547456 doi "https://doi.org/10.1016/j.molcel.2017.06.001" @default.
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