Rheb Regulates Mitophagy Induced by Mitochondrial Energetic Status

Mitophagy has been recently described as a mechanism of elimination of damaged organelles. Although the regulation of the amount of mitochondria is a core issue concerning cellular energy homeostasis, the relationship between mitochondrial degradation and energetic activity has not yet been considered. Here, we report that the stimulation of mitochondrial oxidative phosphorylation enhances mitochondrial renewal by increasing its degradation rate. Upon high oxidative phosphorylation activity, we found that the small GTPase Rheb is recruited to the mitochondrial outer membrane. This mitochondrial localization of Rheb promotes mitophagy through a physical interaction with the mitochondrial autophagic receptor Nix and the autophagosomal protein LC3-II. Thus, Rheb-dependent mitophagy contributes to the maintenance of optimal mitochondrial energy production. Our data suggest that mitochondrial degradation contributes to a bulk renewal of the organelle in order to prevent mitochondrial aging and to maintain the efficiency of oxidative phosphorylation. Mitophagy has been recently described as a mechanism of elimination of damaged organelles. Although the regulation of the amount of mitochondria is a core issue concerning cellular energy homeostasis, the relationship between mitochondrial degradation and energetic activity has not yet been considered. Here, we report that the stimulation of mitochondrial oxidative phosphorylation enhances mitochondrial renewal by increasing its degradation rate. Upon high oxidative phosphorylation activity, we found that the small GTPase Rheb is recruited to the mitochondrial outer membrane. This mitochondrial localization of Rheb promotes mitophagy through a physical interaction with the mitochondrial autophagic receptor Nix and the autophagosomal protein LC3-II. Thus, Rheb-dependent mitophagy contributes to the maintenance of optimal mitochondrial energy production. Our data suggest that mitochondrial degradation contributes to a bulk renewal of the organelle in order to prevent mitochondrial aging and to maintain the efficiency of oxidative phosphorylation. High OXPHOS activity enhances mitochondrial degradation by mitophagy Rheb controls mitochondrial energetic status-induced mitophagy Rheb interacts with Nix and LC3-II to promote mitophagy Mitochondrial energetic status-induced mitophagy preserves OXPHOS efficiency The regulation of mitochondrial content is essential for cellular energetic homeostasis, and this regulation is finely tuned according to cellular energy demand and supply. The cellular mitochondrial content is determined by two complex mechanisms: mitochondrial biogenesis and degradation. Mitochondrial biogenesis has been thoroughly investigated, and its importance in terms of energy homeostasis is well established (Scarpulla, 2011Scarpulla R.C. Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network.Biochim. Biophys. Acta. 2011; 1813: 1269-1278Crossref PubMed Scopus (845) Google Scholar). In contrast, the link between bioenergetics and mitochondrial degradation remains unclear. The degradation of cytosolic components such as organelles can occur through autophagy. Mitophagy is a specific form of autophagy in which mitochondria are specifically targeted for autophagic degradation by the lysosomes (Lemasters, 2005Lemasters J.J. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging.Rejuvenation Res. 2005; 8: 3-5Crossref PubMed Scopus (920) Google Scholar). Three distinct mechanisms of mitophagy have been characterized in the last decade (Youle and Narendra, 2011Youle R.J. Narendra D.P. Mechanisms of mitophagy.Nat. Rev. Mol. Cell Biol. 2011; 12: 9-14Crossref PubMed Scopus (2221) Google Scholar). The first mechanism has been elucidated in yeast and implicates the mitochondrial protein Atg32 as the mitochondrial receptor for the vacuole targeting of mitochondria to mitophagy. This mechanism is regulated by another mitochondria protein, Uth1 (Kissová et al., 2004Kissová I. Deffieu M. Manon S. Camougrand N. Uth1p is involved in the autophagic degradation of mitochondria.J. Biol. Chem. 2004; 279: 39068-39074Crossref PubMed Scopus (346) Google Scholar; Okamoto et al., 2009Okamoto K. Kondo-Okamoto N. Ohsumi Y. A landmark protein essential for mitophagy: Atg32 recruits the autophagic machinery to mitochondria.Autophagy. 2009; 5: 1203-1205Crossref PubMed Scopus (43) Google Scholar). The second mitophagic mechanism has been observed during erythrocyte maturation (Sandoval et al., 2008Sandoval H. Thiagarajan P. Dasgupta S.K. Schumacher A. Prchal J.T. Chen M. Wang J. Essential role for Nix in autophagic maturation of erythroid cells.Nature. 2008; 454: 232-235Crossref PubMed Scopus (870) Google Scholar). This mechanism involves the outer mitochondrial membrane receptor Nix (or Bnip3l) and autophagosome-associated protein LC3 (Novak et al., 2010Novak I. Kirkin V. McEwan D.G. Zhang J. Wild P. Rozenknop A. Rogov V. Löhr F. Popovic D. Occhipinti A. et al.Nix is a selective autophagy receptor for mitochondrial clearance.EMBO Rep. 2010; 11: 45-51Crossref PubMed Scopus (911) Google Scholar). The third mechanism involves the Parkinson’s disease-related proteins Pink and Parkin (Narendra et al., 2008Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J. Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2842) Google Scholar). Parkin is recruited to the mitochondria when the mitochondrial membrane potential (ΔΨ) is abrogated by uncoupling, thereby promoting mitophagy (Narendra et al., 2008Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J. Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2842) Google Scholar). Although these mechanisms of mitophagy represent significant advances in our understanding of mitochondrial turnover, the two mitophagic mechanisms described in mammalian cells appear to be restricted to specific physiological conditions. The Pink/Parkin pathway facilitates the clearance of damaged mitochondria after acute stress, whereas mitophagy in reticulocytes is restricted to a specific developmental stage. Yet early studies have proposed the existence of a basal degradation mechanism that contributes to mitochondrial turnover. It has been shown that mitochondria may be completely renewed within 14 days in different tissues and cell types (Huemer et al., 1971Huemer R.P. Lee K.D. Reeves A.E. Bickert C. Mitochondrial studies in senescent mice. II. Specific activity, buoyant density, and turnover of mitochondrial DNA.Exp. Gerontol. 1971; 6: 327-334Crossref PubMed Scopus (31) Google Scholar; Menzies and Gold, 1971Menzies R.A. Gold P.H. The turnover of mitochondria in a variety of tissues of young adult and aged rats.J. Biol. Chem. 1971; 246: 2425-2429Abstract Full Text PDF PubMed Google Scholar). This renewal requires both mitochondrial biogenesis and degradation. However, the specific mechanism of degradation that participates in the turnover remains to be determined. The cellular mitochondrial energetic status elicits a powerful control over major mitochondrial processes, such as mitochondrial dynamics (Benard et al., 2007Benard G. Bellance N. James D. Parrone P. Fernandez H. Letellier T. Rossignol R. Mitochondrial bioenergetics and structural network organization.J. Cell Sci. 2007; 120: 838-848Crossref PubMed Scopus (489) Google Scholar), mitochondrial biogenesis (Scarpulla, 2008Scarpulla R.C. Transcriptional paradigms in mammalian mitochondrial biogenesis and function.Physiol. Rev. 2008; 88: 611-638Crossref PubMed Scopus (1138) Google Scholar), and calcium signaling (Bianchi et al., 2004Bianchi K. Rimessi A. Prandini A. Szabadkai G. Rizzuto R. Calcium and mitochondria: mechanisms and functions of a troubled relationship.Biochim. Biophys. Acta. 2004; 1742: 119-131Crossref PubMed Scopus (114) Google Scholar). Thus, mitochondrial bioenergetics may also be linked to mitophagy, and several studies have identified a relationship between mitophagy and mitochondrial bioenergetic parameters using either pharmacological treatments (Chen et al., 2007Chen Y. McMillan-Ward E. Kong J. Israels S.J. Gibson S.B. Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species.J. Cell Sci. 2007; 120: 4155-4166Crossref PubMed Scopus (361) Google Scholar; Jin et al., 2010Jin S.M. Lazarou M. Wang C. Kane L.A. Narendra D.P. Youle R.J. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL.J. Cell Biol. 2010; 191: 933-942Crossref PubMed Scopus (883) Google Scholar; Narendra et al., 2008Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J. Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2842) Google Scholar) or oxidative phosphorylation (OXPHOS) yeast mutants (Graef and Nunnari, 2011Graef M. Nunnari J. Mitochondria regulate autophagy by conserved signalling pathways.EMBO J. 2011; 30: 2101-2114Crossref PubMed Scopus (149) Google Scholar; Priault et al., 2005Priault M. Salin B. Schaeffer J. Vallette F.M. di Rago J.P. Martinou J.C. Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast.Cell Death Differ. 2005; 12: 1613-1621Crossref PubMed Scopus (241) Google Scholar). Nevertheless, the link between mitochondrial energetic activity and a physiological degradation of mitochondria is still unknown. In this study, we demonstrated that the stimulation of mitochondrial energy metabolism can regulate mitophagy. We present molecular evidence showing that the small GTPase Rheb (Ras homolog enriched in brain protein) is recruited to the mitochondrial outer membrane upon high OXPHOS activity and regulates mitochondrial energetic status-induced mitophagy. We propose that Rheb participates in the regulation of the renewal of mitochondria to maintain a constant efficiency of mitochondrial energy production. To better understand the link between mitochondrial activity and mitochondrial degradation, we analyzed mitochondrial degradation during conditions of high and low OXPHOS activity. To increase this activity, we switched HeLa cells from media containing glucose and no glutamine to glucose-free media supplemented with glutamine, as previously reported (Reitzer et al., 1979Reitzer L.J. Wice B.M. Kennell D. Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells.J. Biol. Chem. 1979; 254: 2669-2676Abstract Full Text PDF PubMed Google Scholar; Rossignol et al., 2004Rossignol R. Gilkerson R. Aggeler R. Yamagata K. Remington S.J. Capaldi R.A. Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells.Cancer Res. 2004; 64: 985-993Crossref PubMed Scopus (609) Google Scholar; Weinberg et al., 2010Weinberg F. Hamanaka R. Wheaton W.W. Weinberg S. Joseph J. Lopez M. Kalyanaraman B. Mutlu G.M. Budinger G.R. Chandel N.S. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity.Proc. Natl. Acad. Sci. USA. 2010; 107: 8788-8793Crossref PubMed Scopus (1184) Google Scholar). In the same way, human primary skeletal muscle myoblasts (HSMMs) were cultured in the presence or absence of glutamine to have, respectively, low or high OXPHOS activity. Under glutamine-supplemented conditions, HeLa cells displayed a 2-fold increase in the routine oxygen consumption rate (OCR) compared to the cells grown in the presence of glucose only (Figure 1A). The noncoupled OCR induced by the addition of oligomycin was not different between both conditions, while the maximal OCR induced by FCCP in oxidative conditions was twice the rate observed in the glycolytic condition (Figure 1A). Similar results showing that glutamine increased mitochondria respiration were obtained for HSMM (see Figure S1A online). Under routine steady state, HeLa cells produced equivalent amounts of ATP irrespective of the presence of glutamine or glucose. However, the cells grown under oxidative conditions exhibited a strong decrease in mitochondrial ATP production when treated with the complex I inhibitor rotenone, whereas the cells grown in glycolytic conditions were insensitive to the drug (Figure 1B). Using different mitochondrial markers, we could exclude the possibility that this raise was not due to an increase of total mitochondrial content but corresponded to a better feeding of energy substrate to the OXPHOS (Figure S1C). Then we examined the rate of protein degradation by treating both HeLa and HSMM cells with the protein synthesis inhibitor, cycloheximide (CHX). As a result, cells with high OXPHOS activity (grown in glutamine media) revealed a higher rate of mitochondrial protein degradation compared to low OXPHOS activity (cells grown in glucose). Impact of CHX treatment on protein levels of other cellular compartments such as endoplasmic reticulum (calregulin), Golgi (Golgi-58), lysosome (Lamp2), or cytosol (Actin, Gapdh) were independent of energetic conditions and cell types (Figures 1C–1F). This suggests that solely mitochondrial protein degradation rate is impacted by OXPHOS status. We hypothesized that this degradation of mitochondrial proteins occurred via mitophagy. Thus, we examined mitophagy using a combination of microscopy, immunoblot, and pharmacological inhibition analyses. First, we analyzed the microtubule-associated protein light chain 3 (LC3), which is conjugated to phosphatidylethanolamine (namely, LC3-II) and targeted to autophagic membranes upon the induction of autophagy. In HeLa cells, we observed that a gradual increase of the glutamine concentration promoted the conjugation of LC3-II (Figure 2A) along with an increase in the OXPHOS activity (Figure S1B). Conversely, the switch from oxidative to glycolytic metabolism reversed the LC3-II activation within 4 hr, showing that this phenomenon was inducible and reversible (Figure 2B). We also observed an increase in the number of lysosomes in the cells grown in glutamine compared to glucose (Figures S2A and S2B). To test whether a mitophagic process was responsible for the degradation of mitochondria upon OXPHOS activation, we analyzed the effect of autophagic inhibition on the mitochondrial degradation. The inhibition of autophagic/lysosomal degradation using protease inhibitors pepstatin/E64D or by silencing the autophagic protein 7 (ATG7) hampered mitochondrial degradation induced in glutamine condition (Figures 2C and 2D). The same results were obtained using other autophagic inhibitors (Figures S2C and S2D). Imaging of cells expressing GFP-LC3 by immunofluorescence microscopy showed LC3 mainly cytosolic in cells grown in glucose but localized as punctate structures in cells grown in glutamine (Figure 2E). Image analysis revealed that the cells grown in glutamine showed a 4-fold increase in the number of LC3 foci per cell compared to cells grown in glucose (Figure 2F). Furthermore, live imaging of GFP-LC3-transfected cells revealed that the newly formed autophagosomes (green foci) could sequentially tether and engulf the mitochondria in glutamine conditions (Figure 2G and Figure S2E). All together, these data suggested that an increase in OXPHOS activity is accompanied by increased mitophagy. Mitochondrial production of energy via glutamine is mediated by the TCA cycle after the oxidation of glutamine to α-ketoglutarate. To test whether glutamine triggers mitophagy by stimulating mitochondrial energy metabolism through the TCA cycle, we pharmacologically inhibited or bypassed glutaminolysis and assessed the level of LC3-II. First, cells in glutamine media were supplemented with 7 mM dimethyl α-ketoglutarate (DMKG), a cell-permeable analog of α-ketoglutarate, in order to bypass glutaminolysis. Glucose was also added in the media in order to avoid starvation stress. Under these conditions, the ratio of LC3-II to total LC3 significantly increased from 0.43 ± 0.09 to 0.67 ± 0.13 (Figure 3A). In contrast, the inhibition of glutamine catabolism using 2 mM aminooxyacetic acid (AOAA) inhibited mitochondrial respiration and did not promote LC3 activation, as indicated by the ratio of 0.34 ± 0.07 (Figures 3A and 3B). This indicated that the sole presence of glutamine was not sufficient to promote LC3-II formation but that glutamine oxidation by TCA cycle was required to induce mitophagy. When the inhibition of glutamine catabolism by AOAA was bypassed by the addition of DMKG, mitochondrial respiration was restored, and the level of LC3-II was again increased with a ratio of 0.78 ± 0.16. Therefore, the catabolism of glutamine triggered the conjugation of LC3-II, which is the step required for mitophagy to proceed. Then, cells were placed into an oxygraph chamber and permeabilized with digitonin, and the respiration was stimulated with sequential addition of succinate, ADP and FCCP. Cells were collected, and the conversion of LC3 was analyzed (Figure 3C). The results show that the level of LC3-II increased relative to the permeabilized control cells as the respiratory chain was stimulated with succinate. The corresponding mitochondrial respiration is reported in Figure 3D. Further increase of the mitochondrial respiration either by stimulation of ATP synthesis with ADP or by uncoupling with FCCP did not produce any further significant increase of LC3-II (Figure 3C). Taken together, these results demonstrated that a high mitochondrial respiratory chain activity elicits a direct regulatory effect on mitophagy. To further study the mechanism underlying the mitochondrial energetic status-induced mitophagy, we analyzed the mRNA expression of several autophagy- or mitophagy-related genes after switching the cells from oxidative to glycolytic conditions. As expected, the expression levels of the essential autophagic genes ATG9 and ATG12, as well as the mitochondrial mitophagic receptor Nix, were decreased under glycolytic conditions relative to high OXPHOS conditions. Interestingly, we also found that the expression of Rheb decreased to 50% when the cells were switched to glycolytic conditions (Figure S3A). To test whether Rheb participated in the regulation of mitochondrial energetics-induced mitophagy, we analyzed the impact of ectopic expression of myc-tagged Rheb (Myc-Rheb). As shown in Figure 4A, the expression of Rheb in cells grown in glutamine decreased the overall amount of mitochondria relative to control-transfected cells, as determined by flow-cytometric analysis of cells stained with Mitotracker. Furthermore, the gradual increase in the transient expression of Rheb over 48 hr in cells grown either in glutamine or glucose revealed that Rheb overexpression readily induced the activation of LC3 (Figure 4B). Likewise, we observed a specific reduction in mitochondrial protein levels in both oxidative and glycolytic cells concomitant with the increase of Rheb expression (Figure 4B). Notably, the expression of Rheb was sufficient to induce mitophagy during glycolytic conditions. We observed the same activation of LC3 and specific decrease of mitochondrial protein content in HSMM (Figure 4C). Rheb involvement in mitophagy was further confirmed by microscopy in HeLa cell expressing GFP-LC3, which showed that Rheb expression alone induced the formation of autophagosomes that partially colocalized with mitochondria in glucose-grown cells (Figure 4D). The overexpression of Rheb induced also the intracellular conversion of endogenous LC3 to LC3-II which colocalized with mitochondria (Figure S3B). Inhibition of protein degradation with the protease inhibitors pepstatin A and E64D blocked autophagic degradation (Figure 4E) and hampered mitochondrial degradation (Figure S3C). In the same manner, blockade of autophagic machinery by ATG7 silencing hampered mitochondrial degradation induced by Rheb (Figure 4F). Interestingly, we observed a strong accumulation of Rheb when ATG7 was silenced, suggesting that Rheb is degraded during this mitophagic process. As shown in Figure 4G, the shRNA-mediated downregulation of Rheb specifically induced strong accumulation of mitochondria in HeLa cells. Using conditional knockout mice, we found that absence of Rheb in liver induced a 2-fold increase of mitochondrial proteins (Figures 4H and 4I). However, OXPHOS activity (namely, NADH dehydrogenase activity) was not increased in the conditional knockout, indicating that this accumulation is dysfunctional (Figure 4J). All together, these findings indicate that Rheb elicits a potent control on mitophagy. Like several other Ras homologs, Rheb is farnesylated at a C-terminal CAAX domain, and this posttranslational modification anchors the protein to various endomembranes. Interestingly, Rheb has been suggested to be recruited to mitochondria (Ma et al., 2008Ma D. Bai X. Guo S. Jiang Y. The switch I region of Rheb is critical for its interaction with FKBP38.J. Biol. Chem. 2008; 283: 25963-25970Crossref PubMed Scopus (50) Google Scholar). To determine whether Rheb is recruited to mitochondria during mitochondrial energetic status-induced mitophagy, we first analyzed its cellular distribution using a whole-cell fractionation method. As shown in Figure 5A, endogenous Rheb was present within the cytosolic fractions (fractions 1 and 2), the lysosomal/autophagosomal fractions (fractions 6 and 7), and the heaviest fractions containing mitochondria (fractions 8 and 10). In the HeLa cells grown in the presence of glutamine, immunofluorescence assays confirmed that Myc-Rheb located strongly to mitochondria (Figure 5B), as tested by the Intensity Correlation Analysis method (Li et al., 2004Li Q. Lau A. Morris T.J. Guo L. Fordyce C.B. Stanley E.F. A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization.J. Neurosci. 2004; 24: 4070-4081Crossref PubMed Scopus (576) Google Scholar). The intensity correlation quotient (ICQ) scale ranges between −0.5 and 0.5 where 0.5 corresponds to 100% colocalization, and this quotient was 0.42 ± 0.01 between Tom20-stained mitochondria and Myc-Rheb (n > 50 cells). Immunofluorescence also revealed that in 81% of the cells (n = 320 cells) exhibited a mitochondrial localization of Rheb under oxidative conditions, and this value dropped to 45% of the cells (n = 420 cells) under glycolytic conditions (Figures 5C). We also compared the distribution of endogenous Rheb according to the energetic status of the cells using cell fractionation. Although Rheb was present in mitochondrial fractions in both oxidative (glutamine-grown) and glycolytic (glucose-grown) cells, Rheb was enriched in the mitochondrial fractions isolated from cells grown in oxidative conditions (Figure 5D). The mitochondrial amount of Rheb normalized to Tom20 was 0.72 ± 0.13 in oxidative cells compared to 0.35 ± 0.10 in glycolytic cells (n = 4; p < 0.05). Mitochondrial localization was also obtained by cell fractionation with cells expressing Myc-Rheb (Figure S3D). A trypsin digestion assay was performed using isolated mitochondria from cells expressing Myc-Rheb to estimate the submitochondrial membrane localization of Rheb. The results showed that the Myc tag in the N terminus region of Rheb was digested in the same manner as the OMM protein Tom20, which suggested the OMM localization of Rheb (Figure 5E). The anchorage of Rheb to the OMM occurs via the farnesyl group, because the cells transfected with a GFP-Rheb-SSVM mutant (i.e., that cannot be farnesylated) did not locate to mitochondria either in glucose (ICQ 0.09 ± 0.02, mito versus GFP-Rheb-SSVM, n > 60 cells) or in glutamine (ICQ 0.11 ± 0.03, n > 60 cells) (Figure 5F). Furthermore, although cells transfected with this mutant displayed LC3 conjugation, mitochondrial degradation was impeded (Figure 5G), showing the importance of the mitochondrial localization of Rheb in this mitophagic mechanism. In summary, Rheb was preferentially recruited to the OMM in glutamine-grown cells with high OXPHOS activity to promote mitophagy. To confirm whether Rheb controls mitochondrial energetic status-induced mitophagy, we first assessed the involvement of the mammalian target of rapamycin complex 1 (mTORC1), since Rheb activates mTORC1 (Sancak et al., 2010Sancak Y. Bar-Peled L. Zoncu R. Markhard A.L. Nada S. Sabatini D.M. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.Cell. 2010; 141: 290-303Abstract Full Text Full Text PDF PubMed Scopus (1689) Google Scholar). The inactivation of mTORC1 during starvation or amino acid deprivation stimulates autophagy (Zoncu et al., 2011Zoncu R. Bar-Peled L. Efeyan A. Wang S. Sancak Y. Sabatini D.M. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase.Science. 2011; 334: 678-683Crossref PubMed Scopus (1150) Google Scholar), and it has been suggested that mTORC1 is also recruited at the OMM (Desai et al., 2002Desai B.N. Myers B.R. Schreiber S.L. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction.Proc. Natl. Acad. Sci. USA. 2002; 99: 4319-4324Crossref PubMed Scopus (220) Google Scholar). As shown in Figure 6A, we found that the activation state of mTORC1 pathways was similar between glycolytic and oxidative conditions. Also, Rheb overexpression further activated the mTORC1 pathways, as reflected by the phosphorylation status of different mTORC1 substrates (Figure 6A). These results indicated that Rheb expression activated mTORC1, demonstrating that this signaling pathway is not involved in the mitophagy induced by the stimulation of mitochondrial bioenergetics. The phosphorylation status of mTORC1 was also maintained under AOAA and DMKG treatment (Figure S4A). Moreover, in oxidative conditions, mTORC1 partially colocalized with Rheb (ICQ = 0.22 ± 0.03), but poor mitochondrial localization was observed (ICQ = 0.10 ± 0.03). Rheb and mitochondria strongly colocalized (ICQ = 0.42 ± 0.01) (Figure 6B). Previous studies showed that Nix is necessary for mitophagy through physical interaction with the autophagosomal protein LC3 (Novak et al., 2010Novak I. Kirkin V. McEwan D.G. Zhang J. Wild P. Rozenknop A. Rogov V. Löhr F. Popovic D. Occhipinti A. et al.Nix is a selective autophagy receptor for mitochondrial clearance.EMBO Rep. 2010; 11: 45-51Crossref PubMed Scopus (911) Google Scholar). Thus, we tested if Nix was involved in mitochondrial energetic status-induced mitophagy through an interaction with Rheb. Interestingly, we found that the silencing of Nix hampered the Rheb-induced mitophagy, leading to a 2-fold increase of mitochondria (Figure 6C and Figure S4B), but it did not inhibit LC3 conjugation (Figure 6C). Using BN-PAGE, we also identified a 300 kDa complex containing Nix, Rheb, and LC3 (Figure S4C), and silencing of Rheb modified the profile of Nix oligomerization (Figure S4D). Also, coimmunoprecipitation assays performed on cells expressing Myc-Rheb showed that Rheb physically interacts with both endogenous Nix and LC3 (Figure 6D). This interaction occurred mainly in the mitochondrial fraction of glutamine-grown cells (Figure 6E).We calculated that the ratio of Nix immunoprecipitated from the mitochondrial fraction over the total amount in the input was equal to 1.25 ± 0.25 in glucose compared to 1.80 ± 0.32 in glutamine (n = 3). In summary, Rheb forms a complex with Nix and LC3 that is required for mitochondrial energetic status-induced mitophagy. We investigated the effect of Rheb-dependent mitophagy on mitochondrial physiology. Mitophagy has been described as a quality control process that eliminates damaged mitochondria. Under basal conditions, mitochondria from glucose- or glutamine-cultured cells presented neither a collapse of the ΔΨ, an increase of oxidized proteins, nor a mitochondrial network fragmentation (data not shown). Yet, in oxidative conditions, mitochondria produced slightly more reactive oxygen species (ROS) than in glycolytic conditions and the treatment with the autophagic inhibitor-stimulated ROS production (Figure S5A). In cells that were grown in glutamine media and treated with chloroquine, the mitochondria exhibited high levels of carbonylated proteins (Figures 7A and 7B ). The treatment of cells with rotenone (complex I inhibitor and ROS inducer) also resulted in an increase in mitochondrial protein carbonylation compared to the control-treated cells, whereas the expression of Rheb reduced the rotenone-induced protein carbonylation by 51.1% ± 12.6% (Figures 7A and 7B). Furthermore, Rheb expression significantly decreased ROS production, while silencing of Rheb had no effect on ROS levels (Figure 7C). These findings strongly suggested that Rheb could ensure the physiological renewal of the mitochondrial pool before the emergence of damaged mitochondria. Therefore, we analyzed whether Rheb had an impact on mitochondrial energy production. We found that the expression of Rheb increased the routine mitochondrial respiration rate by about 25% and enhanced ATP production by 40% (Figures 7D and 7E). Conversely, the silencing of Rheb did not alter the routine respiration, but the maximal oxygen consumption capacity (FCCP-induced OCR) decreased from 72.2 ± 5.6 to 49.8 ± 4.1 nmol/min/million cells (Figure 7F). Also, the loss of Rheb that resulted in an important uncoupling was associated with a decrease in the ATP content (Figure 7G). The downregulation of Rheb and the associated decrease of ATP strongly impeded cellular proliferation in oxidative conditions, but not in glycolytic conditions (Figure S5B). Reconstitution of Rheb-knockout MEFs with human Rheb was sufficient to enh