Mechanistic analysis of mitochondrial fission mediators

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) to the Department of Biochemistry and Genetics, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe Institute for Molecular Sciences, La Trobe University, Bundoora.

Mitochondria are dynamic organelles continuously undergoing fission and fusion. Fission is important for mitochondrial function and is executed by dynamin related protein 1, Drp1. Drp1 is a cytosolic GTPase and requires adaptors at the mitochondrial surface for its mitochondrial recruitment. MiD51, MiD49, Mff and Fis1 have been implicated as Drp1 adaptors. This project investigates the individual role of each adaptor in recruiting Drp1 using a novel approach based on gene editing and proteomics and validates the importance of Mff and MiD proteins in recruitment but not Fis1. The proteomic analysis also reveals that MiD51 and Mff can exist in the same complex with Drp1 and possibly regulate the fission process. To understand the regulation process, MiD51 and Mff were in vitro reconstituted onto liposomes along with Drp1 and various biochemical methods were employed to measure Drp1 activity. The analysis revealed a novel mechanism by which both the adaptors modulate Drp1 activity distinct from each other. MiD51 is involved in inhibiting Drp1 GTPase activity, whereas Mff stimulates it. The results obtained have been integrated to develop a working model analyzing the role of each adaptor in recruitment and assembly of Drp1 at sites of fission. The model describes the role of MiD51 in stimulating assembly of Drp1 dimers to form a functional polymer around the mitochondrial surface and the role of Mff in enhancing constriction of the polymer to ultimately cause fission. The results presented in the thesis indeed provide novel insights into the role of adaptors in Drp1-dependent mitochondrial fission.