AAA Proteases Research Articles

An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins.

FtsH, a AAA protease, associates with HflK/C subunits to form a megadalton complex that spans the inner membrane and extends into the periplasm of E. coli . How this complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following overproduction of protein components, recent cryo-EM structures reveal symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit degradation of membrane-embedded substrates. Here, we present structures of native complexes in which HflK/C instead forms an asymmetric nautilus-like assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. The membrane curvature in our FtsH·HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid-scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.

The structural remodeling of the mitochondrial Ca2+ uniporter complex underlies its functional impairment during metabolic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR Introduction Calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter complex (MCUC) controls key steps of oxidative metabolism to adapt energy production to cardiac demand. To ensure this critical function, MCUC assembly and maturation is a tightly regulated process involving the AAA proteases. We recently demonstrated that mitochondrial Ca2+ uptake is impaired during the early stages of metabolic cardiomyopathy in the atria. Purpose Our aim was to investigate mitochondrial Ca2+ handling in the left ventricle during the early stages of metabolic cardiomyopathy and to characterize the mechanisms underlying the functional remodeling of the MCUC. Methods Male C57Bl6/J mice were fed a high-fat sucrose diet (HFS) for two weeks to induce metabolic cardiomyopathy. Mitochondrial Ca2+ uptake was then studied (1) in isolated ventricular cardiomyocytes using whole-cell patch-clamp coupled with confocal microscopy and (2) in isolated mitochondria using Ca2+ Green-5N. The biophysical properties of MCUC were recorded in a planar lipid bilayer. MCUC structure and AAA-proteases expression were assessed by Western blot and co-immunoprecipitation. Control mice were treated with AAV9 harboring shRNA to downregulate the expression of MAIP1. Results Metabolic cardiomyopathy was associated with reduced beat-to-beat mitochondrial Ca2+ uptake in ventricular cardiomyocytes, which was further confirmed in isolated mitochondria. Single-channel recordings of the native cardiac MCUC embedded in a planar lipid bilayer revealed a reduction in ionic conductance and open probability, demonstrating functional impairment of the MCUC. Biochemical analysis of the MCUC revealed an accumulation of the unprocessed form of the EMRE subunit and a decreased interaction of the EMRE-MICU1 subcomplex with the pore-forming MCU subunits, indicating a structural remodeling of the MCUC. The expression of the AAA proteases YME1L and AFG3L2/SPG7, which are responsible for EMRE maturation and insertion into the MCUC, was unchanged. However, the expression of m-AAA protease-interacting protein 1 (MAIP1), which orchestrates EMRE processing, was reduced. In control mice, shRNA-mediated downregulation of MAIP1 recapitulated the structural and functional changes of the MCUC. Conclusions The structural remodeling of the MCUC integrity by downregulation of its assembly affects mitochondrial Ca2+ homeostasis in the early stages of metabolic cardiomyopathy. MAIP1 is a key regulator of MCUC assembly and its downregulation underlies the functional remodeling of the MCUC. These findings provide a novel post-translational paradigm of MCUC regulation in pathophysiology and suggest novel therapeutic targets for the treatment of the metabolic cardiomyopathy.

Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca2+ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2.

Cerebral ischemia-reperfusion (I/R) injury as the consequence of revascularization after ischemic stroke is associated with mitochondrial dysfunction, oxidative stress, and neuron loss. In this study, we used a deprivation/reoxygenation (OGD/R) model to determine whether interactions between Netrin-1, AKT, and the mitochondrial AAA protease AFG3L2 could influence mitochondrial function in neurons after I/R. We found that Netrin-1 protects primary cortical neurons from OGD/R-induced cell death and regulates mitochondrial reactive oxygen species (ROS) and Ca2+ levels. The accumulation of mitochondrial calcium uniporter (MCU) subunits was monitored in cells by immunoblot analysis. Although the regulatory subunits MICU1 and MICU2 were relatively unaffected, the accumulation of the essential MCU regulator (EMRE) subunit was impaired. In OGD/R-induced cells, the 7kDa form of EMRE was significantly reduced. Netrin-1 inhibited the accumulation of EMRE and mitochondrial Ca2+ levels by upregulating AFG3L2 and AKT activation. Loss of AFG3L2 or inhibition of AKT increased levels of 7kDa EMRE. Moreover, overexpression of AKT increased the expression of AFG3L2 in Netrin-1-knockdown neurons after OGD/R. Our results demonstrate that Netrin-1 enhanced AFG3L2 protein expression via activation of AKT. We also observed that overexpression of Netrin-1 significantly reduced infarction size in an I/R-induced brain injury model in rats but not when AKT was inhibited. Our data suggest that AFG3L2 is a protein substrate of AKT and indicate that Netrin-1 attenuates cerebral I/R injury by limiting mitochondrial ROS and Ca2+ levels through activating AKT phosphorylation and AFG3L2.

Mitochondrial AAA protease gene associated with immune infiltration is a prognostic biomarker in human ovarian cancer.

Early diagnosis and identification of prognostic markers for ovarian cancer can significantly improve survival and reduce mortality. The role of the YME1L1 signaling axis in genetic alterations and immune infiltration of the tumor microenvironment remains unclear. Bioinformatics web resources, including GEPIA2, cBioPortal, Oncomine, Kaplan-Meier Plotter, and TIMER, were used to analyze the expression profile, prognostic value, and immune infiltration of YME1L1. We further performed a tissue microarray analysis of paraffin-embedded tissues from 60 patients with ovarian cancer, recorded at the FIGO/TNM cancer staging. Here, we used multi-omics analysis of multiple histological datasets to map the role of epigenetic and genetic alterations of YME1L1 in tumor immune infiltration and the prognosis of cancer patients. We explored YME1L1 gene expression profiles by systematically analyzing the association between YME1L1 expression and the prognosis of patients with ovarian cancer confirmed in multiple databases. High expression levels of YME1L1 were associated with poor overall and disease-free survival. Together, our studies suggest that YME1L1 may modulate tumor survival and immune features and contribute to tumor immune invasion, poor prognosis, and immunotherapy failure. Our findings may have clinical implications for the design of treatment strategies, prognosis assessment, and follow-up management of patients receiving immunotherapy for various cancers.

Genome-wide identification and expression analysis of the ftsH protein family and its response to abiotic stress in Nicotiana tabacum L

BackgroundThe filamentous temperature-sensitive H protease (ftsH) gene family plays an important role in plant growth and development. FtsH proteins belong to the AAA protease family. Studies have shown that it is a key gene for plant chloroplast development and photosynthesis regulation. In addition, the ftsH gene is also involved in plant response to stress. At present, the research and analysis of the ftsH gene family are conducted in microorganisms such as Escherichia coli and Oenococcus and various plants such as Arabidopsis, pear, rice, and corn. However, analysis reports on ftsH genes from tobacco (Nicotiana tabacum L.), an important model plant, are still lacking. Since ftsH genes regulate plant growth and development, it has become necessary to systematically study this gene in an economically important plant like tobacco.ResultsThis is the first study to analyze the ftsH gene from Nicotiana tabacum L. K326 (NtftsH). We identified 20 ftsH genes from the whole genome sequence, renamed them according to their chromosomal locations, and divided them into eight subfamilies. These 20 NtftsH genes were unevenly distributed across the 24 chromosomes. We found four pairs of fragment duplications. We further investigated the collinearity between these genes and related genes in five other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified differential expression patterns of NtftsH in different tissues and under various abiotic stress conditions.ConclusionsThis study provides a comprehensive analysis of the NtftsH gene family. The exon–intron structure and motif composition are highly similar in NtftsH genes that belong to the same evolutionary tree branch. Homology analysis and phylogenetic comparison of ftsH genes from several different plants provide valuable clues for studying the evolutionary characteristics of NtftsH genes. The NtftsH genes play important roles in plant growth and development, revealed by their expression levels in different tissues as well as under different stress conditions. Gene expression and phylogenetic analyses will provide the basis for the functional analysis of NtftsH genes. These results provide a valuable resource for a better understanding of the biological role of the ftsH genes in the tobacco plant.

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the β2-β3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and invivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.

Reprogramming of mitochondria by proteolysis

Mitochondria are central metabolic hubs whose structure and function dynamically adapt to changing metabolic demands and environmental challenges. Metabolic reprogramming of mitochondria occurs during development, cell differentiation, in ageing and disease and is coupled to changes in mitochondrial mass and shape. Similarly, stress conditions and altered metabolic cues induce mitochondrial adaptations, altering proteostasis and lipid homeostasis of mitochondria. ATP-dependent AAA proteases residing in the inner membrane of mitochondria are emerging as central regulators of these adaptive responses.

FtsH is required for protein secretion homeostasis and full bacterial virulence in Edwardsiella piscicida

Edwardsiella piscicida, as an important pathogen of fish, has caused huge losses in aquaculture. The virulence in E. piscicida has been increasingly concerned, but few studies have focused on the relationship between virulence and protein secretion homeostasis. FtsH, as a member of the AAA protease family, has important cellular functions, such as controlling the quality of membrane proteins. In this study, FtsH was demonstrated to be essential in maintaining protein secretion homeostasis, and its deletion could result in the secretion of massive cytoplasmic proteins by non-classical secretion pathway. Furthermore it was showed that FtsH is vital for E. piscicida to proliferate within host cells, and E. piscicida mutant ΔftsH will be obviously attenuated. After zebrafish was infected with the mutant ΔftsH, the lethality rate for zebrafish and the bacterial colonization in its organs were greatly reduced. These results suggested that FtsH, as a regulatory factor, closely linked protein secretory homeostasis with bacterial virulence, which provided clues for further exploring the involvement of protein secretion homeostasis in bacterial virulence.

Quality control of protein import into mitochondria.

Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria and deliver non-imported proteins to proteasomal degradation. Second, quality control factors remove translocation stalled precursor proteins from protein translocases. Third, protein translocases monitor protein sorting to mitochondrial subcompartments. Fourth, AAA proteases of the mitochondrial subcompartments remove mislocalized or unassembled proteins. Finally, impaired efficiency of protein transport is an important sensor for mitochondrial dysfunction and causes the induction of cellular stress responses, which could eventually result in the removal of the defective mitochondria by mitophagy. In this review, we summarize our current understanding of quality control mechanisms that govern mitochondrial protein transport.

The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly.

ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidizing NADH and transferring electrons to the ubiquinone pool. Typically, Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP-dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named as rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterized Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

Regulation of mitochondrial plasticity by the i-AAA protease YME1L.

Mitochondria are multifaceted metabolic organelles and adapt dynamically to various developmental transitions and environmental challenges. The metabolic flexibility of mitochondria is provided by alterations in the mitochondrial proteome and is tightly coupled to changes in the shape of mitochondria. Mitochondrial proteases are emerging as important posttranslational regulators of mitochondrial plasticity. The i-AAA protease YME1L, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, coordinates mitochondrial biogenesis and dynamics with the metabolic output of mitochondria. mTORC1-dependent lipid signaling drives proteolytic rewiring of mitochondria by YME1L. While the tissue-specific loss of YME1L in mice is associated with heart failure, disturbed eye development, and axonal degeneration in the spinal cord, YME1L activity supports growth of pancreatic ductal adenocarcinoma cells. YME1L thus represents a key regulatory protease determining mitochondrial plasticity and metabolic reprogramming and is emerging as a promising therapeutic target.

Mutations in the m-AAA proteases AFG3L2 and SPG7 are causing isolated dominant optic atrophy.

ObjectiveTo improve the genetic diagnosis of dominant optic atrophy (DOA), the most frequently inherited optic nerve disease, and infer genotype-phenotype correlations.MethodsExonic sequences of 22 genes were screened by new-generation sequencing in patients with DOA who were investigated for ophthalmology, neurology, and brain MRI.ResultsWe identified 7 and 8 new heterozygous pathogenic variants in SPG7 and AFG3L2. Both genes encode for mitochondrial matricial AAA (m-AAA) proteases, initially involved in recessive hereditary spastic paraplegia type 7 (HSP7) and dominant spinocerebellar ataxia 28 (SCA28), respectively. Notably, variants in AFG3L2 that result in DOA are located in different domains to those reported in SCA28, which likely explains the lack of clinical overlap between these 2 phenotypic manifestations. In comparison, the SPG7 variants identified in DOA are interspersed among those responsible for HSP7 in which optic neuropathy has previously been reported.ConclusionsOur results position SPG7 and AFG3L2 as candidate genes to be screened in DOA and indicate that regulation of mitochondrial protein homeostasis and maturation by m-AAA proteases are crucial for the maintenance of optic nerve physiology.

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane‐bound ATP‐dependent processive metalloprotease FtsH and cleaves MgtE, the major high‐affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+/Zn2+ toxicity. The N‐terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER‐associated degradation (ERAD). Conceptually, the YqgP‐FtsH system we describe here is analogous to a primordial form of “ERAD” in bacteria and exemplifies an ancestral function of rhomboid‐superfamily proteins.

Down-regulation of the mitochondrial i-AAA protease Yme1L induces muscle atrophy via FoxO3a and myostatin activation.

Muscle atrophy is closely associated with many diseases, including diabetes and cardiac failure. Growing evidence has shown that mitochondrial dysfunction is related to muscle atrophy; however, the underlying mechanisms are still unclear. To elucidate how mitochondrial dysfunction causes muscle atrophy, we used hindlimb‐immobilized mice. Mitochondrial function is optimized by balancing mitochondrial dynamics, and we observed that this balance shifted towards mitochondrial fission and that MuRF1 and atrogin‐1 expression levels were elevated in these mice. We also found that the expression of yeast mitochondrial escape 1‐like ATPase (Yme1L), a mitochondrial AAA protease was significantly reduced both in hindlimb‐immobilized mice and carbonyl cyanide m‐chlorophenylhydrazone (CCCP)‐treated C2C12 myotubes. When Yme1L was depleted in myotubes, the short form of optic atrophy 1 (Opa1) accumulated, leading to mitochondrial fragmentation. Moreover, a loss of Yme1L, but not of LonP1, activated AMPK and FoxO3a and concomitantly increased MuRF1 in C2C12 myotubes. Intriguingly, the expression of myostatin, a myokine responsible for muscle protein degradation, was significantly increased by the transient knock‐down of Yme1L. Taken together, our results suggest that a deficiency in Yme1L and the consequential imbalance in mitochondrial dynamics result in the activation of FoxO3a and myostatin, which contribute to the pathological state of muscle atrophy.

Astrocyte-specific deletion of the mitochondrial m-AAA protease reveals glial contribution to neurodegeneration.

Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m‐AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m‐AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic‐ataxia, epilepsy and premature death. Here, we investigate the role of Afg3l2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte‐specific deletion of Afg3l2 in the mouse leads to late‐onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and Afg3l2, and therefore, are totally depleted of the m‐AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte‐specific deficiency of Afg3l1 and Afg3l2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non‐cell‐autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death.

Mitochondria organize the cellular proteostatic response and promote cellular senescence.

Mitochondria have relatively independent protein quality control systems, including their own chaperones for protein folding and AAA proteases for protein degradation. Accumulating evidence has shown that cytosolic proteins and disease-causing misfolded proteins can be translocated into mitochondria and then impinge upon their function. It is important to understand the interplay between cellular proteostasis and mitochondria, as impaired proteostasis and mitochondrial dysfunction are causally linked with aging and age-related disorders. This review highlights our recent finding showing that the outer mitochondrial membrane protein FUNDC1, a previously reported mitophagy receptor, interacts with the chaperone protein HSC70 to mediate the mitochondrial translocation of cytosolic proteasomal substrates via the TOM/TIM complex into the mitochondrial matrix where they can be degraded by LONP1 protease. Excessive accumulation of unfolded proteins within mitochondria triggers the formation of Mitochondrion-Associated Protein Aggregates (MAPAs), which are subsequently autophagically degraded in a FUNDC1-dependent manner. We suggest that mitochondria actively organize the cellular proteostatic response and that the interaction between FUNDC1 and HSC70 may represent a new link between impaired proteostasis, mitochondrial dysfunction and cellular aging.

AAA Proteases: Guardians of Mitochondrial Function and Homeostasis.

Mitochondria are dynamic, semi-autonomous organelles that execute numerous life-sustaining tasks in eukaryotic cells. Functioning of mitochondria depends on the adequate action of versatile proteinaceous machineries. Fine-tuning of mitochondrial activity in response to cellular needs involves continuous remodeling of organellar proteome. This process not only includes modulation of various biogenetic pathways, but also the removal of superfluous proteins by adenosine triphosphate (ATP)-driven proteolytic machineries. Accordingly, all mitochondrial sub-compartments are under persistent surveillance of ATP-dependent proteases. Particularly important are highly conserved two inner mitochondrial membrane-bound metalloproteases known as m-AAA and i-AAA (ATPases associated with diverse cellular activities), whose mis-functioning may lead to impaired organellar function and consequently to development of severe diseases. Herein, we discuss the current knowledge of yeast, mammalian, and plant AAA proteases and their implications in mitochondrial function and homeostasis maintenance.

M-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics.

The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m)-AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m-AAA and i-AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper.

The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component.

Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i-AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i-AAA protease in the regulation of mitochondrial biogenesis in plants.

Mitochondrial inner-membrane protease Yme1 degrades outer-membrane proteins Tom22 and Om45.

Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin-proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1-Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo-cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.