Toncheva, Viktoriya Stefanova; (2023) Integration of mitochondrial dynamics, biogenesis and energy provision via the Miro GTPases and MRPL20. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mitochondrial positioning is crucial for meeting local metabolic demands within the cell and, thus, the distribution of mitochondria is tightly regulated. Healthy mitochondria are trafficked in short-distance actin-based and long-range microtubule-dependent trafficking events. The Miro GTPases modulate mitochondrial motility though interactions with the trafficking complexes that couple the kinesin-1, dynein and Myo19 motor proteins to mitochondria. In mammals, there are two Miro paralogues, Miro1 and Miro2. In this thesis, a low respiratory capacity and low levels of subunits of the OXPHOS complexes are observed in cells lacking both Miro proteins. These results led to a search for a possible link between the Miro proteins and the mitochondrial respiratory machinery and guided the identification of the mitochondrial ribosomal protein MRPL20 as an interactor of both Miro1 and Miro2 by yeast two-hybrid screen and by coimmunoprecipitation. Both Miro proteins affect the targeting, total and mitochondrial protein levels of MRPL20 when studied by immunofluorescence and western blotting approaches in wild-type and Miro1/2 double knock-out mouse embryonic fibroblasts. Surprisingly, the absence of both Miro1 and Miro2 causes not only a decrease in total MRPL20 but also the localisation and accumulation of immature, non-cleaved MRPL20 to the nucleoli. The subcellular targeting of MRPL20 depend on the EF-hand and the GTPase1 domains of Miro, which are also crucial for the ability of Miro to adjust mitochondrial positioning in response to physiological changes within the cell. The GTPase1 domain of Miro also regulates the interaction with MRPL20. Along with its N-terminal mitochondrial targeting domain, MRPL20 relies on its C-terminal Miro-binding domain for its proper localisation. Functionally, MRPL20 can alter trafficking through its Miro-binding domain in a Miro-dependent way. Therefore, the interaction between MRPL20 and the Miro proteins provides a mechanism for integration of mitochondrial trafficking, biogenesis and respiration.

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