Characterization of Mitochondrial YME1L Protease Oxidative Stress-Induced Conformational State

Oxidative stress is a common challenge to mitochondrial function where reactive oxygen species are capable of significant organelle damage. The generation of mitochondrial reactive oxygen species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle, respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged, misfolded, or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal, transmembrane, AAA + (ATPases associated with various cellular activities), and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ± 2 and 26 ± 1 mM for conditions lacking or including nucleotide, respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria, but does stimulate a 2-fold increase in the rate constant for high-affinity ATP binding from (8.9 ± 0.2) × 10⁵ M⁻¹ s⁻¹ to (1.5 ± 0.1) × 10⁶ M⁻¹ s⁻¹. Taken together, these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.     

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS‐dependent proteolysis. Enzymes containing oxidation‐sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress‐dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP‐dependent protease Pim1/LON as a major factor in the degradation of ROS‐modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.     

The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i‐AAA protease YME1L

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring‐like structures and locally specify the protein–lipid composition in a variety of cellular membranes. Stomatin‐like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i‐AAA protease YME1L, which we term the SPY complex (for SLP2–PARL–YME1L). Association with SLP2 in the SPY complex regulates PARL‐mediated processing of PTEN‐induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress‐activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1‐mediated processing of the dynamin‐like GTPase OPA1 allowing stress‐induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.     

YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.     

Mitochondrial adaptation in obesity is a ClpPicated business

Quality control systems that maintain mitochondrial oxidative phosphorylation (OXPHOS) include rescue by mitochondrial fusion, elimination of dysfunctional mitochondria by mitophagy, and degradation of damaged proteins by proteases. ClpP is an ATP‐dependent protease located in the mitochondrial matrix and mutated in Perrault syndrome, causing gonadal atrophy and hearing loss. Given that hearing loss is common in mitochondrial diseases caused by mtDNA mutations, ClpP was proposed to be part of the quality control system to maintain proper mitochondrial OXPHOS function. Two recent studies independently report that deletion of ClpP in mice protects from insulin resistance and obesity by increasing mitochondrial OXPHOS capacity and browning in gonadal white adipose tissue and mitochondrial coupling in brown adipose tissue 1 , 2 . Furthermore, liver‐ and muscle‐specific deletion of ClpP has no major effects on insulin resistance. These studies reveal that ClpP might be involved in tissue‐specific mitochondrial remodeling in response to metabolic demands, rather than exclusively removing damaged proteins to maintain OXPHOS capacity.     

Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential

Background information. Human OPA1 (optic atrophy type 1) is a dynamin‐related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. Results. We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L‐OPA1 (long isoforms of OPA1) and three S‐OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L‐OPA1 to S‐OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy‐metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins‐associated rhomboid‐like protein) – the human orthologue of Pcp1/Rbd1 – and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix‐oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock‐down of YME1L (human yme1‐like protein), an IMS‐oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of ΔΨm (inner mitochondrial membrane potential) or OPA1 processing. Conclusions. Metalloprotease‐mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.     

The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1

Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long‐ and branched‐chain fatty acids for subsequent β‐oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross‐linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging‐related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging‐related diseases indicating that peroxisome maintenance is a critical component of ‘healthy aging’. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age‐dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction.     

Biochemical and functional analysis of the YME1 gene product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae.

Inactivation of YME1 in yeast causes several distinct phenotypes: an increased rate of DNA escape from mitochondria, temperature-sensitive growth on nonfermentable carbon sources, extremely slow growth when mitochondrial DNA is completely absent from the cell, and altered morphology of the mitochondrial compartment. The protein encoded by YME1, Yme1p, contains two highly conserved sequence elements, one implicated in the binding and hydrolysis of ATP, and the second characteristic of active site residues found in neutral, zinc-dependent proteases. Both the putative ATPase and zinc-dependent protease elements are necessary for the function of Yme1p as genes having mutations in critical residues of either of these motifs are unable to suppress any of the phenotypes exhibited by yme1 deletion strains. Yme1p co-fractionates with proteins associated with the mitochondrial inner membrane, is tightly associated with this membrane, and is oriented with the bulk of the protein facing the matrix. Unassembled subunit II of cytochrome oxidase is stabilized in yme1 yeast strains. The data support a model in which Yme1p is an ATP and zinc-dependent protease associated with the matrix side of the inner mitochondrial membrane. Subunit II of cytochrome oxidase, when not assembled into a higher order complex, is a likely substrate of Yme1p.     

Expression of the Saccharomyces cerevisiae gene YME1 in the petite-negative yeast Schizosaccharomyces pombe converts it to petite-positive

Organisms that can grow without mitochondrial DNA are referred to as "petite-positive" and those that are inviable in the absence of mitochondrial DNA are termed "petite-negative." The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the alpha- and gamma-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F(1) component of mitochondrial ATP synthase are also petite-negative. Thus, the F(1) complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.     

Mitochondrial degradation in acetic acid‐induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier

We have previously shown that acetic acid activates a mitochondria‐dependent death process in Saccharomyces cerevisiae and that the ADP/ATP carrier (AAC) is required for mitochondrial outer membrane permeabilization and cytochrome c release. Mitochondrial fragmentation and degradation have also been shown in response to this death stimulus. Herein, we show that autophagy is not active in cells undergoing acetic acid‐induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we found that the vacuolar protease Pep4p and the AAC proteins have a role in mitochondrial degradation using yeast genetic approaches. Depletion and overexpression of Pep4p, an orthologue of human cathepsin D, delays and enhances mitochondrial degradation respectively. Moreover, Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment. AAC‐deleted cells also show a decrease in mitochondrial degradation in response to acetic acid and are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast programmed cell death.     

Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging

Although environmental stress likely plays a significant role in promoting aging, the relationship remains poorly understood. To characterize this interaction in a more comprehensive manner, we examined the stress response profiles for 46 long‐lived yeast mutant strains across four different stress conditions (oxidative, ER, DNA damage, and thermal), grouping genes based on their associated stress response profiles. Unexpectedly, cells lacking the mitochondrial AAA protease gene AFG3 clustered strongly with long‐lived strains lacking cytosolic ribosomal proteins of the large subunit. Similar to these ribosomal protein mutants, afg3Δ cells show reduced cytoplasmic mRNA translation, enhanced resistance to tunicamycin that is independent of the ER unfolded protein response, and Sir2‐independent but Gcn4‐dependent lifespan extension. These data demonstrate an unexpected link between a mitochondrial protease, cytoplasmic mRNA translation, and aging.     

A stress-responsive system for mitochondrial protein degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.