Characterization of the first Mitochondrial SUMO E3 Ligase, MAPL

Abstract

Mitochondrial dynamics play essential roles in a number of physiological processes, including metabolism, the cell cycle, cell stresses and migration. In these instances, mitochondrial morphology, distribution, and function are tightly coordinated by signalling cascades. However, it has been unclear how cellular signalling cascades intersect with the mitochondrial machinery. Many signalling switches utilize post-translational modifications to dynamically alter function, and we have shown that the division rates of mitochondria can be regulated by the conjugation of Drp1 with the Small Ubiquitin Like Modifier 1, SUMO1 (1). This modification functions in concert with phosphorylation events, making Drp1 a central candidate for the regulation of mitochondrial dynamics through signaling pathways. In the first aim of this thesis, I demonstrated that MAPL is a novel mitochondrial SUMO E3 ligase, which modifies Drp1 as well as other mitochondrial substrates. In order to understand the global impact of mitochondrial SUMOylation on mitochondrial fission and other processes, the second objective of this manuscript was to identify the binding partners and substrates of MAPL. To this end, I performed an affinity column which successfully identified a number of MAPL interacting partners, and using a large-scale SUMOylation assay on isolated mitochondria, I have characterized the mitochondrial SUMO proteome. One interacting partner of MAPL identified using affinity chromatography was Vps35, a component of the retromer coat complex. In the third objective of this thesis, I examined whether Vps35 regulates the formation of a novel aspect of mitochondrial dynamics: Mitochondrial Derived Vesicles, MDVs. Our lab has recently shown that MAPL is incorporated in small vesicular profiles that are targeted to peroxisomes. These vesicular profiles contain selected proteins and are formed independently of Drp1, the known mitochondrial fission GTPase. I have shown that the retromer complex is required for the transport of MAPL MDVs to peroxisomes, providing new insights into the mechanism of MDV formation. The work presented in this thesis makes important and novel contributions to our understanding of the mechanisms of mitochondrial SUMOylation, and in establishing the role of MDVs in cellular biology.