Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and ...merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.
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.
From mitochondrial quality control pathways to the regulation of specific functions, the Ubiquitin Proteasome System (UPS) could be compared to a Swiss knife without which mitochondria could not ...maintain its integrity in the cell. Here, we review the mechanisms that the UPS employs to regulate mitochondrial function and efficiency. For this purpose, we depict how Ubiquitin and the Proteasome participate in diverse quality control pathways that safeguard entry into the mitochondrial compartment. A focus is then achieved on the UPS-mediated control of the yeast mitofusin Fzo1 which provides insights into the complex regulation of this particular protein in mitochondrial fusion. We ultimately dissect the mechanisms by which the UPS controls the degradation of mitochondria by autophagy in both mammalian and yeast systems. This organization should offer a useful overview of this abundant but fascinating literature on the crosstalks between mitochondria and the UPS.
•We review how the UPS regulates mitochondrial function and efficiency.•Diverse UPS quality control pathways that safeguard entry into the mitochondria.•UPS-mediated control of the yeast mitofusin Fzo1 in mitochondrial fusion and above.•UPS-mediated control of mitophagy in both mammalian and yeast systems.
Mitochondria are dynamic organelles that undergo permanent fission and fusion events. These processes play an essential role in maintaining normal cellular function. In the yeast Saccharomyces ...cerevisiae, the endoplasmic reticulum-mitochondrial encounter structure (ERMES) is a marker of sites of mitochondrial division, but it is also involved in a plethora of other mitochondrial functions. However, it remains unclear how these different functions are regulated. We show here that Mdm34 and Mdm12, 2 components of ERMES, are ubiquitinated by the E3 ligase Rsp5. This ubiquitination is not involved in mitochondrial dynamics or in the distribution and turnover of ERMES. Nevertheless, the ubiquitination of Mdm34 and Mdm12 was required for efficient mitophagy. We thus report here the first identification of ubiquitinated substrates participating in yeast mitophagy.
Mitofusins are large GTPases that trigger fusion of mitochondrial outer membranes. Similarly to the human mitofusin Mfn2, which also tethers mitochondria to the endoplasmic reticulum (ER), the yeast ...mitofusin Fzo1 stimulates contacts between Peroxisomes and Mitochondria when overexpressed. Yet, the physiological significance and function of these "PerMit" contacts remain unknown. Here, we demonstrate that Fzo1 naturally localizes to peroxisomes and promotes PerMit contacts in physiological conditions. These contacts are regulated through co-modulation of Fzo1 levels by the ubiquitin-proteasome system (UPS) and by the desaturation status of fatty acids (FAs). Contacts decrease under low FA desaturation but reach a maximum during high FA desaturation. High-throughput genetic screening combined with high-resolution cellular imaging reveal that Fzo1-mediated PerMit contacts favor the transit of peroxisomal citrate into mitochondria. In turn, citrate enters the TCA cycle to stimulate the mitochondrial membrane potential and maintain efficient mitochondrial fusion upon high FA desaturation. These findings thus unravel a mechanism by which inter-organelle contacts safeguard mitochondrial fusion.
Mitochondrial integrity relies on homotypic fusion between adjacent outer membranes, which is mediated by large GTPases called mitofusins. The regulation of this process remains nonetheless elusive. ...Here, we report a crosstalk between the ubiquitin protease Ubp2 and the ubiquitin ligases Mdm30 and Rsp5 that modulates mitochondrial fusion. Ubp2 is an antagonist of Rsp5, which promotes synthesis of the fatty acids desaturase Ole1. We show that Ubp2 also counteracts Mdm30-mediated turnover of the yeast mitofusin Fzo1 and that Mdm30 targets Ubp2 for degradation thereby inducing Rsp5-mediated desaturation of fatty acids. Exogenous desaturated fatty acids inhibit Ubp2 degradation resulting in higher levels of Fzo1 and maintenance of efficient mitochondrial fusion. Our results demonstrate that the Mdm30-Ubp2-Rsp5 crosstalk regulates mitochondrial fusion by coordinating an intricate balance between Fzo1 turnover and the status of fatty acids saturation. This pathway may link outer membrane fusion to lipids homeostasis.
We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following beta-adrenergic stimulation, by carrying out real-time imaging ...of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic beta(1) pharmacology and an EC(50) of 4.5 nm. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the beta(1)-adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.
Mitochondria are key organelles that change their size, shape andmorphology undergoing a series of fusion and fission events, referredas mitochondrial dynamics. Pivotal components mediating the ...fusionand fission process of the outer/inner membrane(s) belong to thedynamin family of GTPases named DRPs. While the ability of DRPs tomediate membrane fission is well documented, the mechanism ofouter-membrane Fusion DRPs, named mitofusins are poorlyunderstood. Recently this deadlock was partially circumvented withsolving the truncated structures of human mitofusins MFN1 andMFN2 1, 2. The crystallographic structure of these mini-MFNsrevealed that its fold is strikingly similar to that of the correspondingportion of BDLP, the Bacterial Dynamin Like Protein from thecyanobacteria Nostoc punctiforme 3. GTP binding by BDLP resultsin a switch from a compact structure to an ‘open’ conformation thatstimulates its oligomerization and promotes its ability to shape themorphology of lipid membranes 3. It is thus likely that mitofusinsundergo similar conformational rearrangements. Our group haverecently demonstrated the importance of the N terminal residues 60to 100, which are neither conserved in BDLP nor MFNs, in yeastmitofusin function 4. Hence, structural analysis of the fragmentscontaining these residues is pivotal to understand the role of Fzo1.Herein, we will report our effort to successfully purify andcrystallize a truncated form of Fzo1. This will provide fundamentalinsights into how Fzo1 mediates yeast mitochondrial fusion and itwill allow us to further investigate the role of the residues 60 to 100.1 Y.-L. Cao et al., MFN1 structures reveal nucleotide-triggereddimerization critical for mitochondrial fusion, Nature, vol. 542(2017) 372–376.2 Y.-J. Li et al., Structural insights of human mitofusin-2 intomitochondrial fusion and CMT2A onset, Nat. Commun., 10 (2019)4914.3 H. H. Low, J. Löwe, A bacterial dynamin-like protein, Nature,444 (2006) 766–769.4 D. De Vecchis, L. Cavellini, M. Baaden, J. Hénin, M. M. Cohen,A. Taly, A membrane-inserted structural model of the yeast mitofusinFzo1, Sci. Rep., 7 (2017) 10217.
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