Investigating the SUMOylaton and deSUMOylation of mitochondrial proteins: CharacteriSing the SUMO2/3 conjugation of mitofusion 2

Mitochondria are dynamic organelles which play an important role in cellular energy supply, calcium signalling, cell cycle progression and response to stress. The regulation of mitochondrial protein interactions, subcellular localisation, activity and stability by the Small Ubiquitin-like Modifier (SUMO),often described as a 'molecular switch', is vital for mitochondrial and cellular health. However, the exact roles of mitochondrial protein SUMOylation are still in need of investigation. The deconjugation of SUMO using SUMO proteases is often used to understand how SUMO regulates a protein or a cellular processes. Expressing SUMO proteases elicits off target effects by deSUMOylating non-mitochondrial proteins, making it difficult to study mitochondrial SUMOylation in isolation. A SUMO protease was designed which is targeted to deSUMOylate mitochondrial proteins in isolation and found that deSUMOylating mitochondrial proteins increased mitochondrial respiration, but reduced levels of the mitochondrial fusion protein, mitofusin 2 (Mfn2). It was shown that Mfn2 is conjugated by SUMO2/3 at lysine residues K192 and K502, and is deSUMOylated by the SUMO protease SENP5. Mutation of these residues to arginine demonstrated they are sites of alternative modification (SAM) with ubiquitin, and that SUMO may regulate the non-degradative polyubiquitylation of Mfn2. Overall, the targeted deSUMOylation of mitochondrial proteins was used to identify a mitochondrial SUMO substrate, and site-directed mutagenesis to determine novel SUMOylation sites which could contribute to uncovering the role SUMO plays in regulating Mfn2 function, mitochondrial dynamics and cellular health.