Highlights d ESCRT-III subunits polymerize sequentially at the membrane driven by the ATPase Vps4 d The ESCRT-III polymerization sequence ends by Did2/Ist1 to catalyze membrane fission d Subunit exchange triggers changes in ESCRT-III polymers shape and properties d Tilt introduction in the polymer-membrane interface mediates filament buckling

Balanced fusion and fission are key for proper function and physiology of mitochondria 1,2 . Remodelling of the mitochondrial inner membrane (IM) is mediated by dynamin-like Mitochondrial genome maintenance 1 protein (Mgm1) in fungi or the related Optic atrophy protein 1 (OPA1) in animals [3][4][5] . Mgm1 is required for the preservation of mitochondrial DNA in yeast 6 , whereas mutations in the OPA1 gene in humans are a common cause for autosomal dominant optic atrophy, a genetic disorder affecting the optical nerve 7,8 . Mgm1 and OPA1 are present in mitochondria as a membrane-integral long (l) form and a short (s) form that is soluble in the intermembrane space. Yeast strains expressing temperaturesensitive mutants of Mgm1 9,10 or mammalian cells devoid of OPA1 display fragmented mitochondria 11,12 , suggesting an important role of Mgm1/OPA1 in IM fusion. Consistently, only the mitochondrial outer membrane (OM), but not the IM, fuses in the absence of functional Mgm1 13,14 . Mgm1 and OPA1 have also been shown to maintain proper cristae architecture 10,14 . For example, OPA1 prevents the release of pro-apoptotic factors by tightening cristae junctions 15 . Finally, s-OPA1 localises to mitochondrial constriction sites, where it presumably promotes mitochondrial fission 16 . How Mgm1/OPA1 perform their diverse functions in membrane fusion, scission, and cristae organisation is at present unknown. Here, we present crystal and electron cryo-tomography (cryo-ET) structures of Chaetomium thermophilum Mgm1. Mgm1 consists of a GTPase domain, a bundle signalling element (BSE) domain, a stalk, and a paddle domain containing a membrane binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates assembly of bent tetramers into helical filaments. Cryo-ET of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1/OPA1 filaments dynamically remodel the mitochondrial IM.We purified and crystallised a truncated s-Mgm1 isoform from the thermophilic fungus Chaetomium thermophilum (from here on Mgm1) (Fig. 1a, Extended Data Fig. 1a, Supplementary Data Fig. 1). Crystals of this construct grown in the absence of nucleotides diffracted to 3.6 Å resolution. The structure was solved by single anomalous dispersion (Extended Data Fig. 1b, c, Extended Data Table 1).The structure of Mgm1 contains four domains: A G domain, a bundle signalling element (BSE) domain, a stalk, and a paddle (Fig. 1a, b). The G domain closely resembles that of human dynamin (Extended Data Fig. 2). An interface across the nucleotide-binding site responsible for G domain dimerisation in the dynamin superfamily (the 'G interface') is highly conserved in Mgm1 (Extended Data Fig. 1e). The adjacent BSE domain consists of three helices derived from different regions of Mgm1 (Fig. 1a, b). The BSE domain contacts the G domain, as in the closed conformation of dynamin [17][18][19] . The M...

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The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.

1ESCRT-III is a ubiquitous complex which catalyzes membrane fission from 2 within membrane necks via an as yet unknown mechanism. Here, we reconstituted in 3 vitro the ESCRT-III complex onto membranes. We show that based on variable affinities 4 between ESCRT-III proteins and the ATPase Vps4, subunits are recruited to the 5 membrane in a Vps4-driven sequence that starts with Snf7 and ends with Did2 and Ist1 6 which, together, form a fission-active subcomplex. Sequential recruitment of ESCRT-III 7 subunits is coupled to membrane remodeling. Binding of Did2 promoted the formation of 8 membrane protrusions which later constricted and underwent fission mediated by the 9 recruitment of Ist1. Overall, our results provide a mechanism to explain how a sequence 10 of ESCRT-III subunits drives membrane deformation and fission. 14 catalyze membrane fission from within membrane necks in all investigated cellular 15 processes requiring this type of fission event (1-16). ESCRT-III polymers are nucleated 16 by several factors. ESCRT-II, which binds ESCRT-III core subunit Vps20, is the 17 canonical one. Besides Vps20, yeast ESCRT-III complex contains three other core 18 subunits: Snf7, which binds Vps20-ESCRT-II, as well as Vps2 and Vps24, which, in 19 tandem, bind Snf7 and recruit the AAA-ATPase Vps4 via their [17][18][19][20][21][22][23][24][25][26]. 20 While all ESCRT-III subunits have MIM-domains, their specific affinities for Vps4 differ 21 widely (22,23,25). Vps4 ATPase activity remodels ESCRT-III by inducing turnover of 22 ESCRT-III subunits, controlling the balance between polymer growth (27) and 23 disassembly (24-26). ESCRT-III core subunits assemble, alone or in various 24 stoichiometries, into single or multiple stranded filaments (24,(28)(29)(30)(31)(32)(33). These filaments 25 usually exhibit high spontaneous curvature, but a low rigidity, which leads to diverse 26 helical shapes like spirals (30,(32)(33)(34), conical spirals (28, 34) and tubular helices (24, 27 35). While the possibility that transitions occur between those shapes is under debate (36) 28 many findings support the notion that Vps4-dependent ESCRT-III remodeling promotes 29 membrane constriction and fission (7,27,37). The most direct evidence is the 30 asymmetric constriction of in vitro formed CHMP2-CHMP3 (mammalian homologs of 31 Vps2 and Vps24) tubular copolymers by Vps4 (38). While attempts to reconstitute fission 32 in vitro with ESCRT-III core subunits provided conflicting results regarding the role of 33 2 Vps4 (39, 40), the most constricted polymers observed with Snf7, Vps2 and Vps24 34 exceed a radius of 10 nm (24,27,30,32,33), far from the theoretical limit of 3 nm 35 required for spontaneous fission (41) and far from the radius of experimentally observed 36 dynamin pre-fission intermediates (42). This suggests that other subunits or mechanisms 37 are at play to reach sufficient constriction for membrane fission. 38 Aside from core ESCRT-III proteins, accessory subunits are thought to play a role 39 specific to subsets of ESCRT-III functio...