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.

Two deubiquitylases act on mitofusin and regulate mitochondrial fusion along independent pathways.

Mitofusins, conserved dynamin-related GTPases in the mitochondrial outer membrane, mediate the fusion of mitochondria. Here, we demonstrate that the activity of the mitofusin Fzo1 is regulated by sequential ubiquitylation at conserved lysine residues and by the deubiquitylases Ubp2 and Ubp12. Ubp2 and Ubp12 recognize distinct ubiquitin chains on Fzo1 that have opposing effects on mitochondrial fusion. Ubp2 removes ubiquitin chains that initiate proteolysis of Fzo1 and inhibit fusion. Ubp12 recognizes ubiquitin chains that stabilize Fzo1 and promote mitochondrial fusion. Self-assembly of dynamin-related GTPases is critical for their function. Ubp12 deubiquitylates Fzo1 only after oligomerization. Moreover, ubiquitylation at one monomer activates ubiquitin chain formation on another monomer. Thus, regulation of mitochondrial fusion involves ubiquitylation of mitofusin at distinct lysine residues, intermolecular crosstalk between mitofusin monomers, and two deubiquitylases that act as regulatory and quality control enzymes.

Interplay between the Ubiquitin Proteasome System and Mitochondria for Protein Homeostasis.

Eukaryotic cells are subdivided into membrane-bound compartments specialized in different cellular functions and requiring dedicated sets of proteins. Although cells developed compartment-specific mechanisms for protein quality control, chaperones and ubiquitin are generally required for maintaining cellular proteostasis. Proteotoxic stress is signalled from one compartment into another to adjust the cellular stress response. Moreover, transport of misfolded proteins between different compartments can buffer local defects in protein quality control. Mitochondria are special organelles in that they possess an own expression, folding and proteolytic machinery, of bacterial origin, which do not have ubiquitin. Nevertheless, the importance of extensive cross-talk between mitochondria and other subcellular compartments is increasingly clear. Here, we will present local quality control mechanisms and discuss how cellular proteostasis is affected by the interplay between mitochondria and the ubiquitin proteasome system.

Mitochondrial Surveillance by Cdc48/p97: MAD vs. Membrane Fusion.

Cdc48/p97 is a ring-shaped, ATP-driven hexameric motor, essential for cellular viability. It specifically unfolds and extracts ubiquitylated proteins from membranes or protein complexes, mostly targeting them for proteolytic degradation by the proteasome. Cdc48/p97 is involved in a multitude of cellular processes, reaching from cell cycle regulation to signal transduction, also participating in growth or death decisions. The role of Cdc48/p97 in endoplasmic reticulum-associated degradation (ERAD), where it extracts proteins targeted for degradation from the ER membrane, has been extensively described. Here, we present the roles of Cdc48/p97 in mitochondrial regulation. We discuss mitochondrial quality control surveillance by Cdc48/p97 in mitochondrial-associated degradation (MAD), highlighting the potential pathologic significance thereof. Furthermore, we present the current knowledge of how Cdc48/p97 regulates mitofusin activity in outer membrane fusion and how this may impact on neurodegeneration.

Dynamic survey of mitochondria by ubiquitin.

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria.

Mechanistic perspective of mitochondrial fusion: tubulation vs. fragmentation.

Mitochondrial fusion is a fundamental process driven by dynamin related GTPase proteins (DRPs), in contrast to the general SNARE-dependence of most cellular fusion events. The DRPs Mfn1/Mfn2/Fzo1 and OPA1/Mgm1 are the key effectors for fusion of the mitochondrial outer and inner membranes, respectively. In order to promote fusion, these two DRPs require post-translational modifications and proteolysis. OPA1/Mgm1 undergoes partial proteolytic processing, which results in a combination between short and long isoforms. In turn, ubiquitylation of mitofusins, after oligomerization and GTP hydrolysis, promotes and positively regulates mitochondrial fusion. In contrast, under conditions of mitochondrial dysfunction, negative regulation by proteolysis on these DRPs results in mitochondrial fragmentation. This occurs by complete processing of OPA1 and via ubiquitylation and degradation of mitofusins. Mitochondrial fragmentation contributes to the elimination of damaged mitochondria by mitophagy, and may play a protective role against Parkinson's disease. Moreover, a link of Mfn2 to Alzheimer's disease is emerging and mutations in Mfn2 or OPA1 cause Charcot-Marie-Tooth type 2A neuropathy or autosomal-dominant optic atrophy. Here, we summarize our current understanding on the molecular mechanisms promoting or inhibiting fusion of mitochondrial membranes, which is essential for cellular survival and disease control. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

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.

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.

Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1.

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 alpha-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.

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.

Role of Mitofusins and Mitophagy in Life or Death Decisions.

Mitochondria entail an incredible dynamism in their morphology, impacting death signaling and selective elimination of the damaged organelles. In turn, by recycling the superfluous or malfunctioning mitochondria, mostly prevalent during aging, mitophagy contributes to maintain a healthy mitochondrial network. Mitofusins locate at the outer mitochondrial membrane and control the plastic behavior of mitochondria, by mediating fusion events. Besides deciding on mitochondrial interconnectivity, mitofusin 2 regulates physical contacts between mitochondria and the endoplasmic reticulum, but also serves as a decisive docking platform for mitophagy and apoptosis effectors. Thus, mitofusins integrate multiple bidirectional inputs from and into mitochondria and ensure proper energetic and metabolic cellular performance. Here, we review the role of mitofusins and mitophagy at the cross-road between life and apoptotic death decisions. Furthermore, we highlight the impact of this interplay on disease, focusing on how mitofusin 2 and mitophagy affect non-alcoholic fatty liver disease.

Dual role of a GTPase conformational switch for membrane fusion by mitofusin ubiquitylation.

Mitochondria are essential organelles whose function is upheld by their dynamic nature. This plasticity is mediated by large dynamin-related GTPases, called mitofusins in the case of fusion between two mitochondrial outer membranes. Fusion requires ubiquitylation, attached to K398 in the yeast mitofusin Fzo1, occurring in atypical and conserved forms. Here, modelling located ubiquitylation to α4 of the GTPase domain, a critical helix in Ras-mediated events. Structure-driven analysis revealed a dual role of K398. First, it is required for GTP-dependent dynamic changes of α4. Indeed, mutations designed to restore the conformational switch, in the absence of K398, rescued wild-type-like ubiquitylation on Fzo1 and allowed fusion. Second, K398 is needed for Fzo1 recognition by the pro-fusion factors Cdc48 and Ubp2. Finally, the atypical ubiquitylation pattern is stringently required bilaterally on both involved mitochondria. In contrast, exchange of the conserved pattern with conventional ubiquitin chains was not sufficient for fusion. In sum, α4 lysines from both small and large GTPases could generally have an electrostatic function for membrane interaction, followed by posttranslational modifications, thus driving membrane fusion events.

Nonredundant roles of mitochondria-associated F-box proteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology in yeast.

Mitochondria constantly fuse and divide to adapt organellar morphology to the cell's ever-changing physiological conditions. Little is known about the molecular mechanisms regulating mitochondrial dynamics. F-box proteins are subunits of both Skp1-Cullin-F-box (SCF) ubiquitin ligases and non-SCF complexes that regulate a large number of cellular processes. Here, we analyzed the roles of two yeast F-box proteins, Mfb1 and Mdm30, in mitochondrial dynamics. Mfb1 is a novel mitochondria-associated F-box protein. Mitochondria in mutants lacking Mfb1 are fusion competent, but they form aberrant aggregates of interconnected tubules. In contrast, mitochondria in mutants lacking Mdm30 are highly fragmented due to a defect in mitochondrial fusion. Fragmented mitochondria are docked but nonfused in Deltamdm30 cells. Mitochondrial fusion is also blocked during sporulation of homozygous diploid mutants lacking Mdm30, leading to a mitochondrial inheritance defect in ascospores. Mfb1 and Mdm30 exert nonredundant functions and likely have different target proteins. Because defects in F-box protein mutants could not be mimicked by depletion of SCF complex and proteasome core subunits, additional yet unknown factors are likely involved in regulating mitochondrial dynamics. We propose that mitochondria-associated F-box proteins Mfb1 and Mdm30 are key components of a complex machinery that regulates mitochondrial dynamics throughout yeast's entire life cycle.

Mitofusins: Disease Gatekeepers and Hubs in Mitochondrial Quality Control by E3 Ligases.

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.

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.

Separation and Visualization of Low Abundant Ubiquitylated Forms.

In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.

Cdc48 regulates a deubiquitylase cascade critical for mitochondrial fusion.

Cdc48/p97, a ubiquitin-selective chaperone, orchestrates the function of E3 ligases and deubiquitylases (DUBs). Here, we identify a new function of Cdc48 in ubiquitin-dependent regulation of mitochondrial dynamics. The DUBs Ubp12 and Ubp2 exert opposing effects on mitochondrial fusion and cleave different ubiquitin chains on the mitofusin Fzo1. We demonstrate that Cdc48 integrates the activities of these two DUBs, which are themselves ubiquitylated. First, Cdc48 promotes proteolysis of Ubp12, stabilizing pro-fusion ubiquitylation on Fzo1. Second, loss of Ubp12 stabilizes Ubp2 and thereby facilitates removal of ubiquitin chains on Fzo1 inhibiting fusion. Thus, Cdc48 synergistically regulates the ubiquitylation status of Fzo1, allowing to control the balance between activation or repression of mitochondrial fusion. In conclusion, we unravel a new cascade of ubiquitylation events, comprising Cdc48 and two DUBs, fine-tuning the fusogenic activity of Fzo1.

Systematic mapping of contact sites reveals tethers and a function for the peroxisome-mitochondria contact.

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 ß-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 shaping cuts.

A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.

The critical cis-acting element required for IMD2 feedback regulation by GDP is a TATA box located 202 nucleotides upstream of the transcription start site.

Guanylic nucleotides are essential cellular players, and the critical enzyme in their tightly regulated synthesis in Saccharomyces cerevisiae is encoded by the IMD2 gene. The transcription of IMD2 is subject to general repression by nutrient limitation through the cis nutrient-sensing element. It is also subject to specific feedback regulation by the end products of the guanylic nucleotide synthesis pathway. The critical cis element for this latter mechanism is the guanine response element (GRE), a TATAATA sequence which is located 202 nucleotides upstream of the transcription initiation site and which functions as the IMD2 TATA box. We show that the GRE functions in conjunction with a 52-nucleotide stretch near the transcription start site. This very unusual promoter structure ensures low, basal expression of IMD2 and the recruitment of TFIID to the GRE in response to guanylic nucleotide limitation.