Small ubiquitin-like modifier (SUMO) covalently modifies the substrates on lysine residues. SUMOylation is generally transient and deconjugation is regulated by SUMO-specific proteases, SENPs. SUMO and ubiquitin can also regulate each other. SUMO targeted ubiquitin ligases (STUbLs) have been identified that recognise SUMO and mediate SUMO-dependent ubiquitination. Recent studies have shown that SUMO plays an essential role in mitochondrial dynamics, modifying cytosolic GTPase dynamin related protein (DRP1) and regulating its interaction with adaptor proteins on mitochondrial outer membrane (MOM). Mitochondrial fission factor (Mff) is an adaptor protein that is both ubiquitinated and SUMOylated with SUMOylation site at K151. Yet, the interplay between these modifications on Mff remains unclear. This report presents my findings that ubiquitin-related Mff turn-over is SUMO-dependent. SENP1 and SENP3 proteases promote Mff degradation and mediate Mff deSUMOylation but do not affect Mff ubiquitination directly. SENP-mediated deSUMOylation of Mff resulted in significant reduction of Mff ubiquitination, indicative of SUMO-dependent ubiquitination. This was then tested on ubiquitin ligase Parkin and SUMO ligase MAPL. Overexpressed Parkin did not show significant affect to Mff ubiquitination, but promoted ubiquitination of non-SUMOylatable Mff mutant K151R, indicating K151 might not be the direct ubiquitination site of Parkin. Parkin is a ubiquitin ligase with no reported SUMO ligase activity, yet Parkin overexpression promoted SUMOylation on Mff, suggesting possible binding site competition between SUMO and Ub. MAPL has been reported to mono-SUMOylate DRP1 and promote DRP1-Mff binding. My data demonstrate that MAPL is also a regulator of Mff SUMOylation and turnover. MAPL knocked-down cells showed marked increase in Mff level and reduction in Mff SUMOylation, and MAPL overexpression greatly promoted mitochondrial degradation and possibly fragmentation, suggesting that Mff is a substrate of MAPL-mediated SUMOylation. To conclude, this project demonstrated the crosstalk of SUMOylation and ubiquitination on Mff and the significance of SUMO in regulating Mff degradation.
Investigating mitochondrial fission factor (Mff) SUMOylation and effects on ubiquitination
Chen, Z. (Author). 12 May 2022
Student thesis: Master's Thesis › Master of Science by Research (MScR)