E4 ubiquitin ligase promotes mitofusin turnover and mitochondrial stress response.

Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.

Docking and stability defects in mitofusin highlight the proteasome as a potential therapeutic target.

Defects in mitochondrial fusion are at the base of many diseases. Mitofusins power membrane-remodeling events via self-interaction and GTP hydrolysis. However, how exactly mitofusins mediate fusion of the outer membrane is still unclear. Structural studies enable tailored design of mitofusin variants, providing valuable tools to dissect this stepwise process. Here, we found that the two cysteines conserved between yeast and mammals are required for mitochondrial fusion, revealing two novel steps of the fusion cycle. C381 is dominantly required for the formation of the trans-tethering complex, before GTP hydrolysis. C805 allows stabilizing the Fzo1 protein and the trans-tethering complex, just prior to membrane fusion. Moreover, proteasomal inhibition rescued Fzo1 C805S levels and membrane fusion, suggesting a possible application for clinically approved drugs. Together, our study provides insights into how assembly or stability defects in mitofusins might cause mitofusin-associated diseases and uncovers potential therapeutic intervention by proteasomal inhibition.

Analysis of Protein Stability by Synthesis Shutoff.

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.

Plasticity in salt bridge allows fusion-competent ubiquitylation of mitofusins and Cdc48 recognition.

Mitofusins are dynamin-related GTPases that drive mitochondrial fusion by sequential events of oligomerization and GTP hydrolysis, followed by their ubiquitylation. Here, we show that fusion requires a trilateral salt bridge at a hinge point of the yeast mitofusin Fzo1, alternatingly forming before and after GTP hydrolysis. Mutations causative of Charcot-Marie-Tooth disease massively map to this hinge point site, underlining the disease relevance of the trilateral salt bridge. A triple charge swap rescues the activity of Fzo1, emphasizing the close coordination of the hinge residues with GTP hydrolysis. Subsequently, ubiquitylation of Fzo1 allows the AAA-ATPase ubiquitin-chaperone Cdc48 to resolve Fzo1 clusters, releasing the dynamin for the next fusion round. Furthermore, cross-complementation within the oligomer unexpectedly revealed ubiquitylated but fusion-incompetent Fzo1 intermediates. However, Cdc48 did not affect the ubiquitylated but fusion-incompetent variants, indicating that Fzo1 ubiquitylation is only controlled after membrane merging. Together, we present an integrated model on how mitochondrial outer membranes fuse, a critical process for their respiratory function but also putatively relevant for therapeutic interventions.