A new pathway for mitochondrial quality control: mitochondrial‐derived vesicles
2014; Springer Nature; Volume: 33; Issue: 19 Linguagem: Inglês
10.15252/embj.201488104
ISSN1460-2075
AutoresAyumu Sugiura, Gian‐Luca McLelland, Edward A. Fon, Heidi M. McBride,
Tópico(s)Autophagy in Disease and Therapy
ResumoReview8 August 2014free access A new pathway for mitochondrial quality control: mitochondrial-derived vesicles Ayumu Sugiura Ayumu Sugiura Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Gian-Luca McLelland Gian-Luca McLelland Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Edward A Fon Edward A Fon Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Heidi M McBride Corresponding Author Heidi M McBride Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Ayumu Sugiura Ayumu Sugiura Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Gian-Luca McLelland Gian-Luca McLelland Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Edward A Fon Edward A Fon Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Heidi M McBride Corresponding Author Heidi M McBride Montreal Neurological Institute, McGill University, Montreal, QC, Canada Search for more papers by this author Author Information Ayumu Sugiura1, Gian-Luca McLelland1, Edward A Fon1 and Heidi M McBride 1 1Montreal Neurological Institute, McGill University, Montreal, QC, Canada *Corresponding author. Tel: +1 514 398 1808; E-mail: [email protected] The EMBO Journal (2014)33:2142-2156https://doi.org/10.15252/embj.201488104 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The last decade has been marked by tremendous progress in our understanding of the cell biology of mitochondria, with the identification of molecules and mechanisms that regulate their fusion, fission, motility, and the architectural transitions within the inner membrane. More importantly, the manipulation of these machineries in tissues has provided links between mitochondrial dynamics and physiology. Indeed, just as the proteins required for fusion and fission were identified, they were quickly linked to both rare and common human diseases. This highlighted the critical importance of this emerging field to medicine, with new hopes of finding drugable targets for numerous pathologies, from neurodegenerative diseases to inflammation and cancer. In the midst of these exciting new discoveries, an unexpected new aspect of mitochondrial cell biology has been uncovered; the generation of small vesicular carriers that transport mitochondrial proteins and lipids to other intracellular organelles. These mitochondrial-derived vesicles (MDVs) were first found to transport a mitochondrial outer membrane protein MAPL to a subpopulation of peroxisomes. However, other MDVs did not target peroxisomes and instead fused with the late endosome, or multivesicular body. The Parkinson's disease-associated proteins Vps35, Parkin, and PINK1 are involved in the biogenesis of a subset of these MDVs, linking this novel trafficking pathway to human disease. In this review, we outline what has been learned about the mechanisms and functional importance of MDV transport and speculate on the greater impact of these pathways in cellular physiology. Introduction Mitochondria are very complex organelles, housing hundreds of biochemical reactions from energy production to amino acid and lipid synthesis, to hormone production. These biochemical reactions involve substrates and products that flow between the many organelles within the cell. Rather than metabolite shuttling through free diffusion mechanisms, there is increasing evidence that direct interorganellar contacts are required. For example, elemental iron uptake into the mitochondria has been shown to require “kiss-and-run” contacts between the endosome and mitochondria (Zhang et al, 2005; Sheftel et al, 2007). Direct contacts between mitochondria and lipid droplets and peroxisomes are thought to facilitate fatty acid transport. The most advanced understanding of these contacts is between the ER and the mitochondria. It has long been known that ER is the source of lipids for mitochondrial biogenesis (Shiao et al, 1995) and that these contacts are important for cellular calcium homeostasis (Rizzuto et al, 1998). More recently, it was discovered that ER wrapping around the mitochondria marks the sites for mitochondrial division. A molecular understanding of these contacts has been advanced through studies in yeast and mammalian models (Csordas et al, 1999; de Brito & Scorrano, 2008; Kornmann et al, 2009) and is reviewed elsewhere. It is now clear that there is extensive biochemical cross talk between organelles, but the mechanisms are only beginning to emerge (Sheftel et al, 2007; Zehmer et al, 2009; Rowland & Voeltz, 2012; Mesmin et al, 2013). This review will outline the emerging role of vesicular transport as another means of interorganellar communication. Mitochondrial-derived vesicles (MDVs) are generated through the selective incorporation of protein cargoes, which can be limited to the outer membrane, or can include outer, inner membrane, and matrix content, as illustrated in Fig 1 (Neuspiel et al, 2008; Soubannier et al, 2012a,b). Ultrastructural analysis revealed their size to be relatively uniform, between 70 and 150 nm, and their scission does not require the established mitochondrial fission GTPase Drp1 (Neuspiel et al, 2008; Soubannier et al, 2012a,b). Two distinct fates were identified for MDVs, with their targeting either to the late endosome/multivesicular body for degradation (Soubannier et al, 2012a), or to a subpopulation of peroxisomes (Neuspiel et al, 2008). Although this area of research is just emerging, this review will outline the molecular details of cargo selection, vesicle formation, and delivery, as well as the established and predicted impact of these pathways in cellular physiology. Figure 1. Summary of MDVs cargo variabilityImmunofluorescent and EM images illustrate the diversity of cargo-selected MDVs. Immunofluorescent staining of Tom20 (an outer membrane protein) and pyruvate dehydrogenase (PDH, matrix protein) reveals a number of cargo-selected vesicular structures lying outside of the mitochondria (top left panels, circles versus arrowheads). Although Tom20 is absent from PDH-positive structures (arrowheads), EM and biochemical experiments confirm that these vesicles are double membrane bound. An example is shown to the left where both membranes are seen within the vesicle emerging from the intact mitochondria [with permission from Soubannier et al (2012b)]. Similar cargo selectivity is seen for MDVs carrying MAPL that target the peroxisomes [top right panel of immunofluorescent images, taken with permission from Neuspiel et al (2008)]. We also observe single membrane MDVs derived from just the outer mitochondrial membrane (EM panel on right side). Bottom electron microscopic pictures show MDVs containing Tom20 labeled by immunogold particles enter the multivesicular body [taken with permission from Soubannier et al (2012a)]. Scale bars in EM pictures represent 100 nm. Download figure Download PowerPoint The selection of cargo for transport Mitochondrial-vesicle transport carries cargo to peroxisomes and lysosomes. Cargo destined for the lysosomes is ultimately degraded (Soubannier et al, 2012a), and in vitro studies have shown enrichment of oxidized proteins within MDVs (Soubannier et al, 2012b). However, the purpose of vesicle delivery to the peroxisomes is unclear (Mohanty & McBride, 2013). Currently, only one protein is known to traffic to the peroxisomes, a membrane anchored protein ligase called MAPL (also called MULAN, MUL1, GIDE and HADES) (Neuspiel et al, 2008; Braschi et al, 2009). With just one known cargo, it is difficult to predict the mechanisms and principles that govern cargo selection. However, we can look to evolution to help generate testable, working hypotheses. We will first consider the mechanisms of cargo selection based on each target destination separately. Cargo selection for transport to lysosomes There are two primary pieces of evidence that contribute to our understanding of the nature of the cargo en route to the late endosome/lysosome. Firstly, MDVs generated in vitro from isolated mitochondria were shown to be enriched in oxidized protein, in a process that was stimulated by mitochondrial stress (Soubannier et al, 2012b). This reconstitution of MDV formation further revealed a selective incorporation of protein cargo based on the nature of the mitochondrial stress induced. For example, the generation of ROS in the reaction with xanthine oxidase/xanthine led to a stimulation of MDVs carrying the outer membrane pore protein VDAC, but generation of ROS within the mitochondria, upon addition of a complex III inhibitor antimycin A, led to MDVs carrying the complex III subunit core2, without any enrichment in VDAC (Soubannier et al, 2012b). These data suggest that potentially any cargo could be included within MDVs; assuming that they are first oxidized, which would “damage” the complex. This also suggests that oxidation may trigger aggregation or oligomerization, acting as a seed to initiate membrane curvature from the inside. The process of MDV formation has likely been conserved from archaebacteria, the mitochondrion's ancestors (Deatherage & Cookson, 2012). So, are there clues as to the cargo selection mechanisms within these ancient systems? All gram-negative bacteria, including Archae strains, shed vesicles, from those living within the soil to infectious strains like Helicobacter pylori, causing ulcers, or Treponema pallidum, the cause of syphilis (Kulp & Kuehn, 2010; Bonnington & Kuehn, 2014). These bacterial strains bud a variety of vesicles carrying specific cargoes, with unique tasks from the transport of virulence factors, or peptides that arrest the host cell cycle, to the generation of a biofilm. In addition, the quantity of bacterial protein incorporation into vesicles ranges from 0.1% to 8–12%, showing a remarkable > 100-fold variation in cargo incorporation (Soubannier et al, 2012b; Bonnington & Kuehn, 2014). Environmental changes in pH or nutrients are often a signal for bacterial vesicle secretion; however, the molecular mechanisms responsible for such varied cargo selection are not well understood (Deatherage & Cookson, 2012). Given the diversity and complexity of bacterial vesicle formation, we consider it unlikely that a single evolutionary set of machinery could be mapped to MDVs from the ancestral mechanisms of shedding. However, the utility of vesicles in all membrane systems has been demonstrated and mitochondria are no exception. Indeed, the lessons from bacteria help to frame our understanding of the use of vesicles as a highly selective way to sort mitochondrial proteins. The second important finding was that the generation of MDVs destined for lysosomes required the protein kinase PINK1 and the cytosolic ubiquitin E3 ligase Parkin (McLelland et al, 2014). PINK1 and Parkin are both mutated in familial cases of Parkinson's disease (PD) (Trinh & Farrer, 2013) and were initially shown to act in a common pathway in mitochondrial quality control in Drosophila models of PD (Clark et al, 2006; Park et al, 2006; Yang et al, 2006). More recent work has shown that PINK1 is targeted to the mitochondria but is normally degraded very rapidly. During import, PINK1 is first cleaved by the matrix processing peptidases and PARL (Jin et al, 2010; Greene et al, 2012); however, almost all of the cleaved PINK1 is then released from the import channel and degraded in the cytosol through the N-end rule proteolytic pathway (Kondapalli et al, 2012; Lazarou et al, 2012; Yamano & Youle, 2013). Upon mitochondrial depolarization, the import machinery is inactivated and PINK1 becomes trapped either within the import channel or becomes anchored to the mitochondrial outer membrane near the import channel (Greene et al, 2012; Lazarou et al, 2012). This exposes the kinase domain to the cytosol where it phosphorylates ubiquitin and Parkin, leading to stable Parkin recruitment and activation at the mitochondrial surface (Kim et al, 2008; Shiba-Fukushima et al, 2012; Iguchi et al, 2013; Kane et al, 2014; Kazlauskaite et al, 2014). Parkin ubiquitinates a series of proteins on the mitochondrial surface, which are then recognized by autophagic adaptor proteins and delivered to the autophagosome (Narendra et al, 2008; Gegg et al, 2010; Lee et al, 2010; Matsuda et al, 2010; Tanaka et al, 2010; Chan et al, 2011; Chen & Dorn, 2013; Sarraf et al, 2013). Given that PINK1 and Parkin are also required for MDV transport, we predict that the same mechanisms apply, but at a much more localized level (see model, Fig 2). The import channels are spatially restricted upon the mitochondrial surface, as shown with super-resolution microscopy or immunoelectron microscopy (Wurm et al, 2011). Protein misfolding in the matrix was recently shown to trigger mitophagy after long incubations (Jin & Youle, 2013), without any loss of electrochemical potential. Therefore, we consider that local protein aggregation at the import site, perhaps due to local oxidative damage, or complex assembly defects, would block the import process. Should the matrix chaperones become saturated, or cardiolipin become oxidized locally, then the inner membrane import channel may fail. Cardiolipin oxidizes to phosphatidic acid, a lipid known to alter membrane curvature (Yurkova et al, 2008; Donaldson, 2009; Horvath & Daum, 2013), and may help initiate the outward bending of the membrane. Upon complete depolarization or organelle dysfunction, the mechanism may switch from a local removal of a “patch” of mitochondrial content to the global arrest of PINK1 in all import channels, activation of the autophagic machinery, and entire engulfment of the organelle. This “patch” may not be strictly cargo selective, since whatever aggregated or oxidized proteins and lipids reside in proximity to an arrested import channel would be ejected. Supporting this concept, the kinetic analysis of events following treatment with antimycin A revealed the generation of MDVs at an early stage of ROS production, while global mitochondrial depolarization led to the kinetically slower process of mitophagy (McLelland et al, 2014). This indicates that MDVs are likely a first round of defense for the mitochondria to eject damaged proteins in order to avoid the complete failure of the organelle. This first response does not require the activation of autophagy machinery, as it occurs in the absence of Atg5, Rab9, or beclin (Soubannier et al, 2012a; McLelland et al, 2014). Figure 2. Working hypothesis for vesicle initiation by PINK1 and Parkin(A) Immunogold staining of endogenous Tom20 within COS7 cells reveals the regular spacing of the import channels indicated by arrowheads. Note the close tethering of three multivesicular bodies to the mitochondria. (B) An illustration of our working hypothesis of PINK1/Parkin-mediated MDV formation. In Step 1, unfolded, oxidized proteins within matrix, triggered by ROS or failure to assemble, leads to protein aggregation (blue). Oxidation of cardiolipin will generate PA, contributing to altered membrane curvature. In Step 2, protein aggregates may saturate chaperones, leading to a very localized failure to import at an individual channel. In addition, local oxidation of cardiolipin would further interfere with import channels. PINK1, which is rapidly imported, would then accumulate at these failed import channels. In Step 3, PINK1 phosphorylates both ubiquitin and the ubiquitin-like domain of Parkin, stabilizing the recruitment of activated Parkin. The ubiquitination activity of Parkin is required to generate MDVs, suggesting that domains on the surface may be cleared. In Step 4, a vesicle is formed and released in a process that will certainly involve a number of unidentified proteins. Future studies are needed to test this hypothesis and uncover the details governing the generation of MDVs. Download figure Download PowerPoint A second argument in favor of the import channel acting as a sentinel for MDV formation is that import channels are restricted to the boundary membranes, where the inner and outer membrane are in close apposition to thread precursor proteins into the matrix and inner membrane. This explains how the two membranes may bud out together, since they would be locked in place by the arrested PINK1 precursor. It also lends insight into why PINK1 and Parkin are not required for the generation of MDVs that carry only outer membrane proteins like the import receptor Tom20. Ultrastructural analysis of mitochondrial single membrane vesicles reveal a more pleotropic appearance, rather like “blebs” than true, well-constructed vesicles (Fig 1, Soubannier et al, 2012b). The trigger for these vesicles may more closely mirror the bacterial mechanisms of outer membrane vesicle release, the mechanisms of which remain unclear. Indeed, despite the relatively greater abundance of these Tom20 outer membrane vesicles compared to, for example, MDVs carrying matrix pyruvate dehydrogenase that lack Tom20, to date no protein machineries required for their biogenesis have been identified. A role for Parkin in vesicle trafficking has been shown previously. In receptor-mediated endocytosis, a ubiquitin-interacting motif (UIM) within the adaptor protein Eps15 was shown to bind the ubiquitin-like domain (UBL) of Parkin (Fallon et al, 2006). This led to the monoubiquitination of Eps15 and inhibition of its capacity to recruit the endocytic machinery, thereby regulating its function as an adaptor for endocytosis of the EGF receptor (EGFR). In this way, by delaying EGFR endocytosis and degradation, Parkin can enhance signaling downstream of the receptor. Parkin was also shown to bind and monoubiquitinate the endocytic BAR domain protein endophilin A via a Ubl-SH3 interaction (Trempe et al, 2009). As BAR domains are involved in membrane remodeling and curvature, this finding further links Parkin to vesicle budding and trafficking machinery. It is unclear what the signal is to recruit and activate Parkin at the cell surface as it is unlikely to be PINK1, which is constitutively targeted to mitochondria. However, very recent data have shown that PINK1, upon stabilization at the mitochondrial outer membrane, phosphorylates ubiquitin at position S65 (Kane et al, 2014; Kazlauskaite et al, 2014; Koyano et al, 2014). Three independent studies demonstrated that phosphorylated ubiquitin efficiently activated Parkin ubiquitin ligase activity at the mitochondrial surface in acute settings of mitochondrial uncoupling. In this situation, there was a nearly stoichiometric phosphorylation of cellular ubiquitin, which is likely to reach other cellular ubiquitin targets. In this way, the generation of phosphor-ubiquitin at the mitochondrial surface could act as a signaling mechanism for a global cellular response to mitochondrial stress. Regardless of the mechanisms by which Parkin is activated in endocytosis, the data indicate that Parkin may have a multifaceted role in vesicle transport, at the plasma membrane, mitochondrial surface, and perhaps elsewhere. It will be particularly interesting to determine whether these or other adapters are involved in the membrane budding and trafficking involved in the biogenesis of the subset of MDVs involving PINK1 and Parkin at mitochondria. Thus, whereas Parkin clearly plays a role in mitophagy, it also has a steady-state role in the removal of selected, oxidized cargo in a pathway parallel to mitophagy. Cargo selection for transport to peroxisomes The fate of mitochondrial cargo transiting to the lysosome is to be degraded. However, it is much less obvious why there may be a need for vesicle transport to the peroxisomes. The only cargo identified to date transits to a subpopulation of peroxisomes, about 10–20% of the total peroxisomes in the cell (Neuspiel et al, 2008; Braschi et al, 2010). Immunogold analysis of MAPL-positive MDVs revealed the presence of two membranes (Neuspiel et al, 2008), leading us to consider that the cargo is not limited to outer membrane content. There is some information on the mechanisms of MAPL enrichment within peroxisome-bound MDVs. The retromer complex containing Vps35, Vps26, and Vps29 was identified as a MAPL binding partner in an affinity chromatography approach (Braschi et al, 2010). The retromer complex was first established as a coat-like complex that binds and enriches cargo into vesicles from the endosome for their return to the Golgi apparatus (Seaman et al, 1998; Arighi et al, 2004; Seaman, 2012). The retromer complex also binds to the sorting nexin family of proteins that contain a PX-BAR domain that facilitates membrane curvature required for vesicle formation. More recent experiments reveal a much broader role for the retromer complex in many transport pathways, where specificity is granted through the combinatorial use of different sorting nexin members, and variants of the retromer subunits (Rojas et al, 2007; Collins et al, 2008; Cullen & Korswagen, 2012). In each case, the Vps35 subunit of the retromer binds to cargo tails, hinting that transport to the peroxisome will be more signal specific compared with the mechanisms of transport to the lysosome. Silencing Vps35 blocked the delivery of MAPL to peroxisomes, confirming the functional requirement for this complex in MDV transport (Seaman, 2012). MAPL contains ubiquitin and SUMO E3 ligase activities within the cytosolic domain (Braschi et al, 2009), however mutations in the RING finger domain did not alter the delivery to the peroxisome (Neuspiel et al, 2008; Braschi et al, 2010), indicating that the SUMOylation/ubiquitination activity of MAPL are not mechanistically required for MDV formation. At this time, we consider that MAPL constitutes vesicle cargo that does not function in the generation of peroxisome-bound vesicles. Clearly, there is a great deal of work remaining to elucidate the extent of cargo incorporation and the role of the retromer complex in this pathway. Vps35 participates in a variety of transport pathways throughout the cell. However, with mutations in Vps35 being recently linked to PD and Alzheimer's disease (Vilarino-Guell et al, 2011; Zimprich et al, 2011), its role in MDV transport has emerged as an intriguing functional arc that may link defects in Vps35 with mitochondrial dysfunction. Future work will determine whether an alteration in cargo delivery to peroxisomes may contribute to PD. Mechanisms of MDV transport and delivery As described above, we have identified three factors required for the generation of at least a subset of MDVs; those carrying matrix content for delivery to the lysosome (PINK1 and Parkin), and MDVs destined for the peroxisome (retromer complex). However, if we look to other vesicle transport paradigms, it is apparent that this is likely the tip of the iceberg. In addition to cargo selection mechanisms, MDV formation will require machineries that facilitate membrane curvature, potential coat complexes, incorporation of fusion machinery, and motility factors. MDVs are formed in the absence of the mitochondrial dynamin GTPase Drp1, indicating additional mechanisms are also required for the final scission event (Neuspiel et al, 2008; Soubannier et al, 2012a; McLelland et al, 2014). The independence of Drp1 is consistent with the diameter of the yeast mitochondrial dynamin (Dnm1) ring limited to 100 nm, which would be too large to constrict an MDV neck (Ingerman et al, 2005). We may find clues as to the identity of MDV factors within the MitoCarta, an annotated map of the mitochondrial proteome (http://www.broadinstitute.org/pubs/MitoCarta/). For example, there are a number of vesicle-related proteins whose roles have not yet been characterized on the mitochondrial outer membrane. Two enzymes are predicted to modulate phosphatidylinositol phosphates (PIPs) on the surface; PI(4)-kinase IIIβ and splice variant of the PI(5)-phosphatase synaptojanin-2A (Nemoto et al, 2001). Although the presence of PIP-based microdomains on the mitochondria has not been studied intensively, PI(3)P domains were observed to form during mitophagy (Yang & Yang, 2013). PIP-related microdomains are known to recruit adaptor proteins that could favor membrane bending and vesicle generation (Mayinger, 2012). Consistent with adaptors that facilitate alterations in membrane curvature, another endophilin family member, endophilin B1 (Karbowski et al, 2004; Takahashi et al, 2005, 2007), and a mitochondrial phospholipase D, MitoPLD (Choi et al, 2006; Huang et al, 2011), may also modulate membrane dynamics at the outer membrane. Endophilin B1 has been implicated in the binding of Bax (Pierrat et al, 2001), beclin (Takahashi et al, 2007) and in the process mitochondrial fission (Karbowski et al, 2004). These lipid binding and modifying enzymes are all candidates for MDV transport machinery given the established roles for their activities in vesicle transport within the biosynthetic and endocytic pathways. Finally, several Rab GTPases have been shown to impact mitochondrial morphology, biogenesis or turnover, including Rab32, Rab11, Rab4, and Rab7 (Alto et al, 2002; Bui et al, 2010; Caza et al, 2013; Landry et al, 2014; Talaber et al, 2014; Yamano et al, 2014). Therefore, it would not be surprising if some of these small GTPases were involved in MDV transport as well. It is also critical to learn how MDVs may fuse with their target organelle. A splice variant of VAMP1A, called VAMP1B, was identified in 1998 and contains a mitochondrial targeting sequence in place of the C-terminal tail anchor (Isenmann et al, 1998). VAMP1B is ubiquitously expressed, whereas VAMP1A variants are exclusive to neurons. The function of VAMP1B is currently unknown, but it is a prime candidate to mediate fusion events of MDVs with target organelles. Mitochondrial proteomic studies have not identified a t-SNARE or SNAP25 homologue, suggesting that the mitochondria may be unable to receive incoming vesicles. Interestingly, a high-resolution proteome of a very divergent mitochondrion-related organelle, called a mitosome, from the parasite Giardia intestinalis was recently published (Jedelsky et al, 2011). Mitosomes have lost their mtDNA, as well as their capacity to respire, and have almost-unrecognizable import machinery. Their major role is in fact to generate iron sulfur clusters for distribution throughout the cell. Despite its divergence from a typical mitochondrion, the highly purified mitosome proteome included potential orthologues of the retromer component Vps35, an R-SNARE 3 (a v-SNARE) and VAP, a VAMP (vesicle associated membrane protein)-interacting protein (Jedelsky et al, 2011). Like mammalian mitochondria, Giardia mitosomes are also limited to v-SNAREs in the absence of t-SNAREs or SNAP orthologues. This provides a clue that even the simplest mitosome may sort cargo within vesicles for delivery within the cell, perhaps to distribute iron sulfur clusters to other organelles, or for degradation as we see in mammalian cells. The physiological contribution of MDV transport to mitochondrial quality control MDV transport to lysosomes adds a fourth mechanism to the paradigms of mitochondrial quality control. MDVs function alongside the actions of mitochondrial proteases, ubiquitin-mediated proteasomal degradation, and mitophagy. The unanswered question is to define the relative contributions and potential hierarchy of these 4 mechanisms (Fig 3). Mitochondrial proteases degrade unfolded and oxidized proteins within the matrix and intermembrane space (Tatsuta & Langer, 2009). In yeast, an in vitro peptide export assay indicated that mitochondrial proteases degrade between 6–12% of proteins per hour, consistent with proteases as a front line of mitochondrial quality control (Augustin et al, 2005). It is also possible that proteases may trim down complexes and cargoes, leaving more hydrophobic regions to be subsequently removed via MDVs, a possible example of the overlap among these pathways. Loss of mitochondrial proteases leads to various forms of neurodegeneration, including spastic paraplegia (Casari et al, 1998; Atorino et al, 2003; Nolden et al, 2005). For example, mutations in AFG3L2, an m-AAA protease within the inner membrane, are responsible for spinocerebellar ataxia 28 (SCA28) (Di Bella et al, 2010). In addition, mitochondrial proteases are required for the processing of PINK1 (Greene et al, 2012), further supporting the interdependence between multiple quality control pathways. Figure 3. Outline of the 4 pathways of mitochondrial quality controlA schematic diagram depicting the presence of mitochondrial proteases within the mitochondrial matrix and intermembrane space, which likely acts as a first line of defense against unfolded and oxidized soluble proteins. Outer membrane proteins are instead removed from the mitochondria through a retrotranslocation pathway following ubiquitination. Degradation of these proteins is completed within the cytosolic proteasome, similar to the ER-associated degradation pathway. We propose that the third line of defense is the removal of mitochondrial patches through the generation of MDVs, which transit to the late endosome. Only upon complete mitochondrial dysfunction, or upon a failure of most import channels would the entire organelle be targeted to the autophagosome. Different tissues and cellular states may rely on each of these mechanisms to a variable degree, m
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