The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in β-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.

Mitochondrial morphology depends on balanced fusion and fission events. A central component of the mitochondrial fusion apparatus is the conserved GTPase Fzo1 in the outer membrane of mitochondria. Mdm30, an F-box protein required for mitochondrial fusion in vegetatively growing cells, affects the cellular Fzo1 concentration in an unknown manner. We demonstrate that mitochondrial fusion requires a tight control of Fzo1 levels, which is ensured by Fzo1 turnover. Mdm30 binds to Fzo1 and, dependent on its F-box, mediates proteolysis of Fzo1. Unexpectedly, degradation occurs along a novel proteolytic pathway not involving ubiquitylation, Skp1–Cdc53–F-box (SCF) E3 ubiquitin ligase complexes, or 26S proteasomes, indicating a novel function of an F-box protein. This contrasts to the ubiquitin- and proteasome-dependent turnover of Fzo1 in α-factor–arrested yeast cells. Our results therefore reveal not only a critical role of Fzo1 degradation for mitochondrial fusion in vegetatively growing cells but also the existence of two distinct proteolytic pathways for the turnover of mitochondrial outer membrane proteins.

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.

Mitochondria are dynamic organelles engaged in quality control and aging processes. They constantly undergo fusion, fission, transport, and anchoring events, which empower mitochondria with a very interactive behavior. The membrane remodeling processes needed for fusion require conserved proteins named mitofusins, MFN1 and MFN2 in mammals and Fzo1 in yeast. They are the first determinants deciding on whether communication and content exchange between different mitochondrial populations should occur. Importantly, each cell possesses hundreds of mitochondria, with a different severity of mitochondrial mutations or dysfunctional proteins, which potentially spread damage to the entire network. Therefore, the degree of their merging capacity critically influences cellular fitness. In turn, the mitochondrial network rapidly and dramatically changes in response to metabolic and environmental cues. Notably, cancer or obesity conditions, and stress experienced by neurons and cardiomyocytes, for example, triggers the downregulation of mitofusins and thus fragmentation of mitochondria. This places mitofusins upfront in sensing and transmitting stress. In fact, mitofusins are almost entirely exposed to the cytoplasm, a topology suitable for a critical relay point in information exchange between mitochondria and their cellular environment. Consistent with their topology, mitofusins are either activated or repressed by cytosolic post-translational modifiers, mainly by ubiquitin. Ubiquitin is a ubiquitous small protein orchestrating multiple quality control pathways, which is covalently attached to lysine residues in its substrates, or in ubiquitin itself. Importantly, from a chain of events also mediated by E1 and E2 enzymes, E3 ligases perform the ultimate and determinant step in substrate choice. Here, we review the ubiquitin E3 ligases that modify mitofusins. Two mitochondrial E3 enzymes—March5 and MUL1—one ligase located to the ER—Gp78—and finally three cytosolic enzymes—MGRN1, HUWE1, and Parkin—were shown to ubiquitylate mitofusins, in response to a variety of cellular inputs. The respective outcomes on mitochondrial morphology, on contact sites to the endoplasmic reticulum and on destructive processes, like mitophagy or apoptosis, are presented. Ultimately, understanding the mechanisms by which E3 ligases and mitofusins sense and bi-directionally signal mitochondria-cytosolic dysfunctions could pave the way for therapeutic approaches in neurodegenerative, cardiovascular, and obesity-linked diseases.