Ayumu Sugiura

A new pathway for mitochondrial quality control: mitochondrial-derived vesicles

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 Parkinsons 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.

MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin-proteasome pathway.

Newly born peroxisomes are a hybrid of mitochondrial and ER-derived pre-peroxisomes.

Peroxisomes function together with mitochondria in a number of essential biochemical pathways, from bile acid synthesis to fatty acid oxidation. Peroxisomes grow and divide from pre-existing organelles, but can also emerge de novo in the cell. The physiological regulation of de novo peroxisome biogenesis remains unclear, and it is thought that peroxisomes emerge from the endoplasmic reticulum in both mammalian and yeast cells. However, in contrast to the yeast system, a number of integral peroxisomal membrane proteins are imported into mitochondria in mammalian cells in the absence of peroxisomes, including Pex3, Pex12, Pex13, Pex14, Pex26, PMP34 and ALDP. Overall, the mitochondrial localization of peroxisomal membrane proteins in mammalian cells has largely been considered a mis-targeting artefact in which de novo biogenesis occurs exclusively from endoplasmic reticulum-targeted peroxins. Here, in following the generation of new peroxisomes within human patient fibroblasts lacking peroxisomes, we show that the essential import receptors Pex3 and Pex14 target mitochondria, where they are selectively released into vesicular pre-peroxisomal structures. Maturation of pre-peroxisomes containing Pex3 and Pex14 requires fusion with endoplasmic reticulum-derived vesicles carrying Pex16, thereby providing full import competence. These findings demonstrate the hybrid nature of newly born peroxisomes, expanding their functional links to mitochondria.

Distinct regulation of mitochondrial localization and stability of two human Sirt5 isoforms

Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5iso1 and SIRT5iso2) encoded by the human SIRT5 gene and whose C‐termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N‐termini, we found that the cleaved SIRT5iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5iso1 was localized in both mitochondria and cytoplasm. SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2. These results suggest that the cytoplasmic localization of cleaved SIRT5iso1 is dependent on the SIRT5iso1‐specific C‐terminus. Further analysis showed that the C‐terminus of SIRT5iso2, which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5iso1‐specific C‐terminus is necessary for maintaining the stability of SIRT5iso1. Moreover, genome sequence analysis from each organism examined indicated that SIRT5iso2 is a primate‐specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate‐specific functions via two isoforms with different intracellular localizations or stabilities.

A mitochondrial ubiquitin ligase MITOL controls cell toxicity of polyglutamine-expanded protein

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (ΔNAT-3Q71) and found that MITOL promoted ΔNAT-3Q71 degradation via the ubiquitin-proteasome pathway and attenuated mitochondrial accumulation of ΔNAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble ΔNAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin-proteasome pathway.

Formation of mitochondrial‐derived vesicles is an active and physiologically relevant mitochondrial quality control process in the cardiac system

Mitochondrial‐derived vesicle (MDV) formation occurs under baseline conditions and is rapidly upregulated in response to stress‐inducing conditions in H9c2 cardiac myoblasts. In mice formation of MDVs occurs readily in the heart under normal healthy conditions while mitophagy is comparatively less prevalent. In response to acute stress induced by doxorubicin, mitochondrial dysfunction develops in the heart, triggering MDV formation and mitophagy. MDV formation is thus active in the cardiac system, where it probably constitutes a baseline housekeeping mechanism and a first line of defence against stress.

Minocycline sensitizes rodent and human liver mitochondria to the permeability transition: Implications for toxicity in liver transplantation

The antibiotic minocycline exerts cytoprotection in animal disease models. One proposed mechanism is modulation of the mitochondrial permeability transition (mPT), a Ca2 -dependent pathogenic event leading to necrotic and/or apoptotic cell death.1–5 A recent study in HEPATOLOGY by Theruvath et al.,6 investigating storage/ reperfusion injury following rat liver transplantation, concluded that minocycline prevented mPT and mitigated liver injury by decreasing mitochondrial Ca2 uptake without affecting mitochondrial respiration. Further, the authors argue that it could be consistent with clinical practice to (pre)treat stored livers and graft recipients with minocycline. The driving force for mitochondrial Ca2 transport is the mitochondrial membrane potential and the amount of Ca2 retained is dependent on the proton gradient and the matrix pH.7 Respiratory inhibition will decrease Ca2 retention capacity and sensitize mitochondria toward mPT.5,7 Further, endogenous inhibitors of mPT such as adenine nucleotides and Mg2 will influence the amount of Ca2 sequestered prior to mPT. In Theruvath et al., the effect of minocyline on mPT was determined in two classical assays, both using bolus additions of calcium chloride: (1) the swelling assay and (2) the Ca2 retention capacity assay. In both assays, the endpoint is Ca2 overload and induction of mPT. The authors found that minocycline prevented Ca2 induced swelling and decreased Ca2 retention and interpreted this as a specific inhibitory effect on Ca2 uptake. They excluded respiratory inhibition as the explanation to their findings by determining the respiration of mitochondria exposed to minocycline with and without Ca2 addition. However, the buffer used in the respiration assay was different from the one used in the Ca2 bolus assays, with high Mg2 concentration (Mg2 is a known endogenous inhibitor of mPT) and with the presence of the potent pharmacological mPT inhibitor cyclosporin A during Ca2 addition. We argue that minocycline at moderate to high dosing, similar to what we have shown in brain mitochondria, prevents Ca2 -uptake and mPT-induced swelling by respiratory inhibition.1,5 Further, depending on the buffer system used, the decreased Ca2 retention can be explained by minocycline-induced increase of mPT sensitivity related to (1) inhibited respiration1,5 and (2) chelating of Mg2 ,8 or (3) direct activation of mPT (even during concurrent cyclosporin A treatment) by adding Ca2 or in Ca2 loaded mitochondria, as recently shown by Kupsch et al.8 To stringently evaluate effects of minocycline during the process of Ca2 uptake, retention, and mPT, mitochondrial oxygen consumption can be monitored during a continuous Ca2 infusion (Fig. 1A,B). This assay provides information of the bioenergetic demand on mitochondria caused by Ca2 uptake as well as the respiratory inhibition triggered by mitochondrial Ca2 overload and mPT.5,7 Alternatively, the effect of minocycline on isolated mitochondria can be displayed by following changes of extramitochondrial Ca2 during a slow infusion of the cation. In these more physiologically relevant models, minocycline dose-dependently reduces Ca2 retention capacity and sensitizes rat and, importantly, human liver mitochondria to the mPT in the dose range used by Theruvath et al. (0-100 nmol/mg mitochondria; Fig. 1). In conclusion, minocycline may be a promising agent for cytoprotection at relevant dosing through mechanisms other than mPT inhibition. In the clinical setting, prevention of mitochondrial Ca2 uptake via respiratory inhibition is likely not beneficial to the organism. Further, to sensitize mitochondria to mPT by chelating Mg2 is not a viable strategy for cytoprotection. This must be kept in mind when considering the use of minocycline, even at moderate dosing, to mitigate storage/reperfusion injury during liver transplantation.