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The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease. The human mitochondrial protease LONP1 is an AAA+ ATP-dependent quality control protease. Here, the authors present the cryo-EM structures of human LONP1 in three distinct states and provide insights into the mechanism and regulation of this important protease.

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.

Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role in controlling mitochondrial function as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel a link between mitochondrial protein quality and muscle mass maintenance during muscle disuse. Mitochondrial function is important for muscle maintenance and function, and mitochondrial proteolysis maintains mitochondrial integrity and function. Here the authors report that that loss of LONP1-dependent mitochondrial proteolysis in muscle causes reduced muscle mass and strength via activation of autophagy.

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG–mtDNA binding, which is antagonized by LONP-1. Yang et al. report that ATFS-1 preferentially accumulates in dysfunctional mitochondria carrying mutated mitochondria DNA (∆mtDNA) and facilitates mtDNA replication by promoting POLG recruitment, resulting in the replicative advantage of ∆mtDNA and heteroplasmy maintenance.

The biogenesis of membrane proteins (MPs) is inherently error-prone, and is therefore monitored by quality control mechanisms that remove faulty MPs. A key challenge for this surveillance is to recognize misfolded MPs, but how this is achieved remains poorly understood. Here we reveal how FtsH, the main MP quality control protease in Escherichia coli , specifically targets faulty MPs. By analyzing the in vivo degradation of two substrates, we show that lipid-facing polar residues trigger FtsH-mediated degradation. In folded MPs, such polar residues are usually buried in the protein core. Their exposure to the membrane can therefore signal misfolding and promote degradation. Strikingly, lipid-facing polar residues can even trigger degradation of a folded protein, and do not require the extended cytosolic regions typically needed for other FtsH substrates. Recognition depends on the FtsH transmembrane domain and on specific polar residues within it. Thus, sensing misfolding within the membrane helps maintain the integrity of the membrane proteome. Misfolded membrane proteins are toxic and must be eliminated from the cell, but how they are recognized remains unclear. Here, the authors show that polar residues facing the hydrophobic membrane act as a degradation cue sensed by E. coli FtsH protease via its transmembrane domain.

Mutations in GBA1 , the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher’s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration. GBA1 mutations cause Gaucher’s disease and are the strongest risk factor for Parkinson’s disease. Using stable cell lines and patient iPSCs, the authors show mitochondrial localization of GBA1, which may affect neurodegenerative disease risk.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli , proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli , complementary to genomics data, that will allow researchers to study the control of proteostasis. Gupta, Johnson et al. quantify the turnover rates of ~3200 E. coli proteins, demonstrating that cytoplasmic proteins are recycled when nitrogen is limited and that protein degradation rates are generally uncoupled from cell division rates.

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding. We show that LONP1, an AAA+ protease of the mitochondrial matrix, works with the mtHSP70 chaperone system to promote mitochondrial protein folding. Inhibition of LONP1 results in aggregation of a protein subset similar to that caused by knockdown of DNAJA3, a co-chaperone of mtHSP70. LONP1 is required for DNAJA3 and mtHSP70 solubility, and its ATPase, but not its protease activity, is required for this function. In vitro, LONP1 shows an intrinsic chaperone-like activity and collaborates with mtHSP70 to stabilize a folding intermediate of OXA1L. Our results identify LONP1 as a critical factor in the mtHSP70 folding pathway and demonstrate its proposed chaperone activity. Most mitochondrial proteins are imported from the cytosol and must fold in the mitochondria. Here, the authors show that the mitochondrial protease LONP1 plays a critical role in the mtHSP70 chaperone system independently of its protease activity.

Mitochondria comprise the central metabolic hub of cells and their imbalance plays a pathogenic role in chronic kidney disease (CKD). Here, we studied Lon protease 1 (LONP1), a major mitochondrial protease, as its role in CKD pathogenesis is unclear. LONP1 expression was decreased in human patients and mice with CKD, and tubular‐specific Lonp1 overexpression mitigated renal injury and mitochondrial dysfunction in two different models of CKD, but these outcomes were aggravated by Lonp1 deletion. These results were confirmed in renal tubular epithelial cells in vitro . Mechanistically, LONP1 downregulation caused mitochondrial accumulation of the LONP1 substrate, 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), which disrupted mitochondrial function and further accelerated CKD progression. Finally, computer‐aided virtual screening was performed, which identified a novel LONP1 activator. Pharmacologically, the LONP1 activator attenuated renal fibrosis and mitochondrial dysfunction. Collectively, these results imply that LONP1 is a promising therapeutic target for treating CKD. Synopsis Activation of the mitochondrial protease Lon protease 1 (LONP1) attenuates mitochondrial dysfunction and renal fibrosis in chronic kidney disease (CKD) mice, suggesting that targeting LONP1 might represent a novel therapeutic strategy for CKD. CKD patients and mice have decreased LONP1 expression. The substrate of LONP1, HMGCS2, accumulates in mitochondria causing mitochondrial dysfunction and accelerating CKD progression. Activation of LONP1 by the newly identified activator 84‐B10 improves mitochondrial function and reduces renal fibrosis in CKD mouse model. Graphical Abstract Activation of the mitochondrial protease Lon protease 1 (LONP1) attenuates mitochondrial dysfunction and renal fibrosis in chronic kidney disease (CKD) mice, suggesting that targeting LONP1 might represent a novel therapeutic strategy for CKD.

Mutations in phosphatase and tensin homologue‐induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD‐linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA‐mediated interference (RNAi)‐based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin‐associated rhomboid‐like protease (PARL), m‐AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1–Parkin pathway. Dysfunctional mitochondria express high surface levels of the Parkinson's disease‐linked protein PINK1, which in turn recruits Parkin for mitophagy. Fon and colleagues now show that levels of PINK1 are kept low in normal mitochondria through degradation by the mitochondrial processing peptidase (MPP).