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Biogenesis of Mitochondrial-Derived Vesicles (MDVs) – Novel Roles of GTPases MIRO1/2 and DRP1

ABSTRACT: MDVs are implicated in diverse physiological processes e.g. mitochondrial quality control and are linked to various neurodegenerative diseases. However, their specific cargo composition and complex molecular biogenesis is still unknown. Here we report for the first time the proteome and lipidome of steady-state TOMM20+-MDVs. We identified 106 novel high confidence MDV cargoes and verified several candidates by super-resolution microscopy including 5 members of the TOM import complex. Additionally, we demonstrate that MDVs deliver fully assembled protein complexes like the TOM complex for lysosomal degradation, thus filling a crucial mitochondrial quality control niche. Moreover, we show key biogenesis steps of MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. We conclude that catalysing scission during MDV biogenesis demonstrates a novel function of DRP1, distinct from its role in mitochondrial division.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Cercopithecus Aethiops (green Monkey) (grivet)

TISSUE(S): Cell Culture

SUBMITTER: Hendrik Nolte

LAB HEAD: Thomas Langer

PROVIDER: PXD023077 | Pride | 2021-09-28

REPOSITORIES: Pride

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Project description:Mitochondria share attributes of vesicular transport with their bacterial ancestors given their ability to form mitochondrial-derived vesicles (MDVs). MDV formation enhances with stress and has been explored for its potential role in mitochondrial-directed cellular and extracellular signalling. Although the fate of MDVs as a form of mitochondrial quality control and cellular communication has been suggested, the proteomic cargo has remained mostly undefined. In this study, we strategically used an in vitro MDV budding/reconstitution assay, following graded oxidative stress on cardiac mitochondria, to identify and characterize the MDV proteome. Our results confirmed previously identified cardiac MDV markers while also revealing a complete map of the MDV proteome, paving the way to understanding the likely roles for MDV release. MDV cargo was particularly enriched with mitochondrial matrix and inner membrane proteins. Oxidative stress vulnerability of proteins directed the cargo loading, which was enhanced by antimycin A (Ant-A). Among OXPHOS complexes, complexes III and V were found to be Ant-A-sensitive. Proteins from metabolic pathways such as TCA cycle, amino acid, fatty acid and aldehyde metabolism, Fe-S cluster, antioxidant response proteins, autophagy and vesicular processes were also found to be Ant-A sensitive. Intriguingly, proteins containing hyper-reactive cysteine residues (HRCR), which act as redox switches in regulating metabolic pathways, including professional redox enzymes and those that are involved in iron metabolism were found to be loaded in MDVs with Ant-A sensitivity. Lastly, we estimated a possible significant contribution of MDVs to the formation of extracellular vesicles, which can serve as an indicator of mitochondrial stress. In conclusion, using graded oxidative stress, our study gives a layout of MDV proteomics signature, delineates the cargo selectivity and hints at the potential of MDVs and the novel protein cargo identified to serve as vital biomarkers during mitochondrial stress and its related pathologies.

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Biogenesis of Mitochondrial-Derived Vesicles (MDVs) – Novel Roles of GTPases MIRO1/2 and DRP1

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