Large GTPases of the Dynamin Related Proteins (DRP) family shape lipid bilayers through membrane fission or fusion processes. Despite the highly organized photosynthetic membranes of thylakoids, a ...single DRP is known to be targeted inside the chloroplast. Fzl from the land plant Arabidopsis thaliana is inserted in the inner envelope and thylakoid membranes to regulate their morphology. Fzl may promote the fusion of thylakoids but this remains to be proven. Moreover, the physiological requirement for fusing thylakoids is currently unknown. Here, we find that the unicellular microalga Chlamydomonas reinhardtii encodes an Fzl ortholog (CrFzl) that is localized in the chloroplast where it is soluble. To explore its function, the CRISPR/Cas9 technology was employed to generate multiple CrFzl knock out strains. Phenotypic analyzes revealed a specific requirement of CrFzl for survival upon light stress. Consistent with this, strong irradiance lead to increased photoinhibition of photosynthesis in mutant cells. Fluorescence and electron microscopy analysis demonstrated that upon exposure to high light, CrFzl mutants show defects in chloroplast morphology but also large cytosolic vacuoles in close contact with the plastid. We further observe that strong irradiance induces an increased recruitment of the DRP to thylakoid membranes. Most importantly, we show that CrFzl is required for the fusion of thylakoids during mating. Together, our results suggest that thylakoids fusion may be necessary for resistance to light stress.
Mitochondria assemble in a highly dynamic network where interconnected tubules evolve in length and size through regulated cycles of fission and fusion of mitochondrial membranes thereby adapting to ...cellular needs. Mitochondrial fusion and fission processes are mediated by specific sets of mechano-chemical large GTPases that belong to the Dynamin-Related Proteins (DRPs) super family. DRPs bind to cognate membranes and auto-oligomerize to drive lipid bilayers remodeling in a nucleotide dependent manner. Although structural characterization and mechanisms of DRPs that mediate membrane fission are well established, the capacity of DRPs to mediate membrane fusion is only emerging. In this review, we discuss the distinct structures and mechanisms of DRPs that trigger the anchoring and fusion of biological membranes with a specific focus on mitofusins that are dedicated to the fusion of mitochondrial outer membranes. In particular, we will highlight oligomeric assemblies of distinct DRPs and confront their mode of action against existing models of mitofusins assemblies with emphasis on recent biochemical, structural and computational reports. As we will see, the literature brings valuable insights into the presumed macro-assemblies mitofusins may form during anchoring and fusion of mitochondrial outer membranes.
•We briefly review the function and architecture of DRPs.•We provide an overview of selected DRP oligomers.•We describe the main properties and architecture of mitofusins.•We discuss how mitofusins may oligomerize.
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.
The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces ...cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome-dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCF(Mdm30p) ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.
Mitochondria undergo frequent fusion and fission events to adapt their morphology to cellular needs. Homotypic docking and fusion of outer mitochondrial membranes are controlled by Mitofusins, a set ...of large membrane-anchored GTPase proteins belonging to the dynamin superfamily. Mitofusins include, in addition to their GTPase and transmembrane domains, two heptad repeat domains, HR1 and HR2. All four regions are crucial for Mitofusin function, but their precise contribution to mitochondrial docking and fusion events has remained elusive until very recently. In this commentary, we first give an overview of the established strategies employed by various protein machineries distinct from Mitofusins to mediate membrane fusion. We then present recent structure-function data on Mitofusins that provide important novel insights into their mode of action in mitochondrial fusion.
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 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.
Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway ...is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.
Mitochondria are dynamic organelles characterized by an ultrastructural organization which is essential in maintaining their quality control and ensuring functional efficiency. The complex ...mitochondrial network is the result of the two ongoing forces of fusion and fission of inner and outer membranes. Understanding the functional details of mitochondrial dynamics is physiologically relevant as perturbations of this delicate equilibrium have critical consequences and involved in several neurological disorders. Molecular actors involved in this process are large GTPases from the dynamin-related protein family. They catalyze nucleotide-dependent membrane remodeling and are widely conserved from bacteria to higher eukaryotes. Although structural characterization of different family members has contributed in understanding molecular mechanisms of mitochondrial dynamics in more detail, the complete structure of some members as well as the precise assembly of functional oligomers remains largely unknown. As increasing structural data become available, the domain modularity across the dynamin superfamily emerged as a foundation for transfering the knowledge towards less characterized members. In this review, we will first provide an overview of the main actors involved in mitochondrial dynamics. We then discuss recent example of computational methodologies for the study of mitofusin oligomers, and present how the usage of integrative modeling in conjunction with biochemical data can be an asset in progressing the still challenging field of membrane dynamics.
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.
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