The mechanism of mitochondrial fission: Dynamin-related protein 1 and its effectors

Abstract

Mitochondrial fission requires the evolutionarily conserved dynamin related GTPase (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane (1--3) to co-ordinate membrane scission (see (4) for review). Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, using yeast two-hybrid, we identify Ubc9 and SUMO1 as novel DRP1 interacting proteins. We have determined that the interaction between DRP1 and SUMO1 requires SUMO1 conjugation and is nucleotide dependent. Pull-down experiments reveal that DRP1 is an authentic SUMO1 substrate and is likely modified by more than one SUMO1 molecule. Biochemical fractionation indicates that purified mitochondrial fractions contain a NEM-sensitive, SDS-resistant high molecular weight form of DRP1, consistent with the size of SUMOylated DRP1. Importantly, fluorescence microscopy reveals that a significant portion of cytosolic YFP:SUMO1 colocalizes with mitochondria. Video analysis further demonstrates that YFP:SUMO1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Surprisingly, immunofluorescence studies show that endogenous DR-P1 only partially colocalizes with the many YFP: SUMO1 puncta seen on the mitochondria, suggesting that mitochondria contain other SUMOylated substrates. This is consistent with the presence of numerous unique SUMOylated products in the mitochondrial fraction. Finally, transient transfection of SUMO1 into cultured cells dramatically increases the level of mitochondrial fragmentation and protects DRP1 from protein degradation. Together, these data are the first to identify a function for SUMO1 on the mitochondria and suggest a novel role for the participation of SUMO1 in mitochondrial fission.