Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion

Dynamin-related GTPase proteins (DRPs) are main players in membrane remodelling. Conserved DRPs called mitofusins (Mfn1/Mfn2/Fzo1) mediate the fusion of mitochondrial outer membranes (OM). OM fusion depends on self-assembly and GTPase activity of mitofusins as well as on two other proteins, Ugo1 and Mdm30. Here, we define distinct steps of the OM fusion cycle using in vitro and in vivo approaches. We demonstrate that yeast Fzo1 assembles into homo-dimers, depending on Ugo1 and on GTP binding to Fzo1. Fzo1 homo-dimers further associate upon formation of mitochondrial contacts, allowing membrane tethering. Subsequent GTP hydrolysis is required for Fzo1 ubiquitylation by the F-box protein Mdm30. Finally, Mdm30-dependent degradation of Fzo1 completes Fzo1 function in OM fusion. Our results thus unravel functions of Ugo1 and Mdm30 at distinct steps during OM fusion and suggest that protein clearance confers a non-cycling mechanism to mitofusins, which is distinct from other cellular membrane fusion events.