ARTICLE https://doi.org/10.1038/s41467-020-15467-7 OPEN A salvage pathway maintains highly functional respiratory complex I ✉ Karolina Szczepanowska 1,2 , Katharina Senft1,2, Juliana Heidler3, Marija Herholz1,2, Alexandra Kukat1,2, Michaela Nicole Höhne4, Eduard Hofsetz1,2, Christina Becker1,2, Sophie Kaspar1,2, Heiko Giese5, Klaus Zwicker 6, Sergio Guerrero-Castillo 7,8, Linda Baumann1,2, Johanna Kauppila9, Anastasia Rumyantseva 1,2, Stefan Müller1, Christian K. Frese1, Ulrich Brandt 7,8, Jan Riemer4, Ilka Wittig3 & ✉ Aleksandra Trifunovic 1,2 1234567890():,; Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability. 1 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany. 2 Institute for Mitochondrial Diseases and Aging, Medical Faculty, University of Cologne, 50931 Cologne, Germany. 3 Functional Proteomics, ZBC, Faculty of Medicine, Goethe University, 60590 Frankfurt am Main, Germany. 4 Department of Chemistry, Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany. 5 Molecular Bioinformatics, Goethe-Universität Frankfurt am Main, 60325 Frankfurt am Main, Germany. 6 Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany. 7 Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands. 8 Cluster of Excellence Frankfurt “Macromolecular Complexes”, Goethe-University, 60590 Frankfurt am Main, Germany. 9 Department of Mitochondrial Biology, Max Planck Institute for Biology of Aging, ✉ 50931 Cologne, Germany. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2020) 11:1643 | https://doi.org/10.1038/s41467-020-15467-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15467-7 itochondria are the central hub for metabolism, pro- presented a much higher incorporation rate than those of the Mviding the majority of cellular ATP yield through the membrane arm, with the N-module subunits being replaced on action of a respiratory chain coupled to the oxidative average six times more, and the Q-module three times more than phosphorylation (OXPHOS). The oxidation of NADH by Com- P-module subunits (Fig. 1a and Supplementary Data 1). plex I (CI, NADH:ubiquinone oxidoreductase), the largest In a recent screen for putative ClpXP substrates, we identified enzyme of the respiratory chain and an essential part of super- several CI peripheral arm components, including all three core complexes, provides the prevailing driving force for ATP subunits of N-module (NDUFV1, NDUFV2, and NDUFS1)18. synthesis1,2.CIdeficiency is the most common OXPHOS ClpXP is multiunit proteasome-like machinery in the mitochon- pathology that has been implicated in the pathogenesis of mito- drial matrix composed of one or two hexamers of the AAA+ chondrial diseases, Parkinsonism, diabetes, cancer, and aging3–5. ATPase CLPX, and a tertadecameric serine protease CLPP19.To Mammalian CI consists of 45 subunits organized in a modular, L- assess whether the CLPP protease is responsible for the rapid shaped, membrane-rooted structure6,7. MtDNA-encoded sub- removal of N-module components, we analyzed the turnover rate units (ND1-ND6, ND4L) together with 21 supernumerary, of newly synthesized NDUFV2 using pulse-chase immunopreci- nuclear-encoded subunits form the membrane arm (P-module) pitation (Fig. 1b). Indeed, we detected a robust stabilization of that allows proton pumping across the inner mitochondrial NDUFV2 upon CLPP depletion, while confirming a rapid membrane6,8,9. The matrix exposed peripheral arm is composed degradation of the protein in the presence of CLPP (Fig. 1b). of 17 nuclear-encoded subunits divided into two functional parts: In the absence of CLPP in cardiomyocytes, N-module subunits a matrix proximal NADH-oxidizing-N-module, and a membrane accumulate in several distinct subcomplexes, the most prominent adjacent ubiquinone-reducing-Q-module. Altogether, they con- of which contains all three, core subunits: NDUFV1, NDUFV2, tain a chain of eight iron-sulfur (FeS) clusters wiring the electron and NDUFS1 (Fig. 1c). In contrast, a putative CLPP substrate flow through the complex1. NDUFS2 and Q-module subunit was not detected in additional The peripheral arm, especially the flavin mononucleotide subcomplexes (Fig. 1c). (FMN) cofactor site in the N-module, is also the primary site of To gain a better insight into assembly intermediates that might CI reactive oxygen species (ROS) production, making it parti- be accumulating in the absence of CLPP, we performed high- cularly prone to intrinsic damage10,11. Thiol-based redox mod- resolution complexome profiling analysis9. A high, 11-fold ifications have been proposed as a ROS-counteracting, self- accumulation of the core N-module subcomplex composed of regulating strategy that transiently reduces electron transfer NDUFV1, NDUFV2, NDUFS1, and NDUFA2 subunits con- within CI to prevent cell and tissue damage12. Therefore, constant firmed our previous analysis (Fig. 1d, Supplementary Fig. 1a and surveillance and replacement of the N-module should be critical Supplementary Data 2). This very robust accumulation of the N- to maintain CI function while preventing extensive oxidative module intermediates cannot be solely explained by augmented damage and might be achieved through matrix quality control CI assembly because other complexes, including the MCIA machinery. complex (mitochondrial CI assembly factor complex), and the Although the principles of stepwise CI biogenesis have been most prominent Q-module subcomplex, showed only a moderate recently described in great detail9, the regulation of its turnover, twofold increase in the CLPP-deficient heart mitochondria. In remodeling, and stability remains enigmatic. Some studies sug- contrast, the amount of P-module assembly intermediates gested that the stability and remodeling of CI might be implicated remained unchanged (Fig. 1e, Supplementary Fig. 1b, and in the regulation of supercomplex formation, response to Supplementary Data 2). Furthermore, we observed a 50% hypoxia, and in a fine-tuning of mitochondrial OXPHOS to the decrease of fully assembled CI, while levels of CIII and CIV available carbon source13–15. While it was proposed that the were much less affected (Fig. 1f, Supplementary Fig. 1a, and membrane-embedded CI components are actively removed by Supplementary Data 2). Consequently, loss of CI in CLPP- iAAA and mAAA proteases16,17, the machinery and mechanisms deficient cardiomyocytes led to decreased levels of supercom- that allow selective clearance of the CI peripheral arm under plexes, while the individual CIII and CIV proportionally physiological condition remain mostly unknown. This is sur- accumulated (Supplementary Fig. 1c and Supplementary Data 2). prising given that the N-module is considered to be the primary Still, putative ClpXP substrates (NDUFV1, NDUFV2, NDUFS1, site of ROS production, and therefore most likely to suffer the NDUFS2, NDUFAB1) showed significantly higher abundance potential ROS-related damage. when compared to other CI subunits (NDUFS4, NDUFS3, or Here, we demonstrate that mitochondrial ClpXP protease is NDUFB11) (Fig. 1g and Supplementary Fig. 1d). required for the turnover of the core part of the N-module of CI, The overall decrease in the amount of assembled CI in the which occurs at a higher rate and largely independently of the rest heart was confirmed by the analysis of all FeS clusters in CI, as of the complex. The regular replacement of the core N-module detected by the EPR (electron paramagnetic resonance) spectro- components is required to prevent the inactivation of CI, which scopy20 in CLPP-deficient mitochondrial membranes (Fig. 1h). In results in defective respiration. Moreover, it acts as a safeguard contrast, the FeS cluster S3, inside complex II, had almost mechanism, which, when OXPHOS is stalled, prevents the elec- identical intensity in both samples (Supplementary Fig. 1e). In tron flow through the respiratory chain, thereby limiting the agreement with the observed loss of CI, and therefore FeS production of ROS. clusters, we detected significant upregulation of several enzymes involved in the synthesis and trafficking of FeS clusters (Supplementary Fig. 1f and Supplementary Data 3). Results N-module subcomplexes accumulate upon CLPP deficiency.To study the CI dynamics, we analyzed protein exchange rates using Subunits of N and P modules