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Membrane constriction and thinning by sequential ESCRT-III polymerization


The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.

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Fig. 1: CHMP1B and IST1 sequentially constrict membrane tubes.
Fig. 2: CHMP1B interlocks in the same arrangement in all structures and flexes at the α3-α4 elbow to accommodate different curvatures.
Fig. 3: IST1 polymerization drives constriction of the CHMP1B + IST1 filament.
Fig. 4: The bilayer thins and the inner leaflet crowds under high curvature in the CHMP1B + IST1 filament.

Data availability

Cryo-EM maps and models were deposited to the PDB and EMDB with the following codes: membrane-bound CHMP1B-only filament (PDB 6TZ9, EMD-20590), membrane-bound right-handed CHMP1B + IST1 filament (PDB 6TZ4, EMD-20588), membrane-bound left-handed CHMP1B + IST1 filament (PDB 6TZ5, EMD-20589), IST1NTDR16E K27E filament (PDB 6TZA, EMD-20591).


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We thank: members of the Frost lab for helpful discussion, especially P. Thomas and M. Sun with computational assistance; M. Grabe, M. Tucker, D. Argudo, E. Lyman, A. Sodt, G. Huber and A. Roux for discussions on membrane biophysical behavior; T. Weiss for assistance with SAXS data collection; M. Braunfeld, D. Bulkley, M. Harrington, A. Myasnikov and Z. Yu of the UCSF Center for Advanced CryoEM for microscopy support; and J. Baker-LePain and the QB3 shared cluster (NIH grant 1S10OD021596-01) for computational support. Structural biology applications used in this project were compiled and configured by SBGrid. The Titan X Pascal used for this research was donated by the NVIDIA Corporation. This work was supported by NSF grant ENG-1563280 (to M.D.V.) and NIH grants P50 AI150464 and 1DP2GM110772-01 (to A.F.), R01 GM112080 and R37 AI51174 (to W.I.S.). A.F. is also supported by a Faculty Scholar grant from the HHMI and is a Chan Zuckerberg Biohub investigator.

Author information




H.C.N. performed biochemical and cryo-EM experiments and analysis. N.T. assisted with cryo-EM experiments and analysis. J.M. contributed to experiments and discussions. A.S. performed optical trap experiments and analysis. F.R.M. performed SAXS experiments and analysis. J.H.I. contributed illustrations and animations as well as discussions. M.D.V., W.I.S. and A.F. supervised all work. H.C.N., W.I.S. and A.F. prepared the manuscript with input from all authors.

Corresponding authors

Correspondence to Wesley I. Sundquist or Adam Frost.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 CryoEM validation of membrane-bound CHMP1B-only filament.

a, Angular distribution of the membrane-bound CHMP1B filament. b, Half map Fourier shell correlation (FSC) of the membrane-bound CHMP1B filament. c, Map to model FSC of the membrane-bound CHMP1B filament.

Extended Data Fig. 2 CryoEM reconstruction of the membrane-bound CHMP1B + IST1 filament at higher curvature and comparison of left- and right-handed CHMP1B + IST1 filaments.

a, CryoEM 3D reconstruction of the membrane-bound left-handed CHMP1B + IST1 filament. End-on view down the helical axis in grey-scale (left) or colored (middle). Right, internal view looking outward from the membrane surface along the helical axis. IST1 protomers (cyan) bind to the exterior of CHMP1B (green), leading to constriction of the membrane (grey). IST1 and CHMP1B protomers are highlighted in dark cyan and green, respectively. Diameters of the entire tube and membrane leaflet peak-to-peak distances are annotated. b, Electron density maps of CHMP1B from the left-handed (left) or right-handed (right) membrane-bound CHMP1B + IST1 filaments. Five copies of CHMP1B are shown as ribbons. c, Superposition of a CHMP1B protomer from the left-handed (purple) and right-handed (green) CHMP1B + IST1 filaments aligned to the CHMP1B N-terminal α1-α2 helices (left) or C-terminal α4-α5 helices (right).

Extended Data Fig. 3 Local resolution estimates and cryoEM validation of membrane-bound CHMP1B + IST1 filaments.

a, Angular distribution of right-handed (top) and left-handed (bottom) membrane-bound CHMP1B + IST1 filaments. b, Half map FSCs of right-handed (top) and left-handed (bottom) CHMP1B + IST1 filaments. c, Map to model FSCs right-handed (top) and left-handed (bottom) CHMP1B + IST1 filaments. d, Local resolution estimates of right-handed (left) and left-handed (right) CHMP1B + IST1 filaments.

Extended Data Fig. 4 IST1 does not discriminate between left- or right-handed CHMP1B filaments.

a, Electron density maps of the IST1 strands from the right-handed (left) or left-handed (right) membrane-bound CHMP1B + IST1 filaments. Two subunits of IST1 are shown as ribbons. b, Superposition of CHMP1B and IST1 protomers from the right-handed (dark green and dark blue, respectively) and left-handed (light green and cyan, respectively) CHMP1B + IST1 filaments. c, Superposition as in (b) but only with the C-terminal region of CHMP1B and IST1. d, Superposition of two subunits of IST1 from the right- or left-handed copolymers from (a).

Extended Data Fig. 5 Real-time monitoring of CHMP1B and IST1 membrane constriction and elongation.

a-c, Still images representing deformation of two membrane tubes due to transverse flow of (a) buffer alone, (b) then 0.5 μM CHMP1B, (c) and a final addition of 0.5 μM IST1. Solid arrows in (a-c) highlight tubule locations. d-e, Contours of lower (d) and upper (e) membrane tubes extracted from panels (a-c) showing the extension of the tubes upon addition of CHMP1B and IST1.

Extended Data Fig. 6 CryoEM validation of the IST1NTDR16E K27E filament.

a, Angular distribution of the IST1NTDR16E K27E filament. b, Half map FSC of the IST1NTDR16E K27E filament. c, Map to model FSC of the IST1NTDR16E K27E filament.

Extended Data Fig. 7 Steric clashing between the CHMP1B MIM and inter-turn IST1 subunits would prevent IST1 from achieving its preferred curvature in the copolymer.

a, External view of the IST1NTDR16E K27E filament with one CHMP1B MIM (shown as a green cylinder) docked onto the IST1NTDR16E K27E j subunit. b, Zoomed in view of boxed area in (a) highlighting how the CHMP1B MIM clashes with the IST1 j + 14 subunit.

Extended Data Fig. 8 Subtle deformations in the outer leaflet observed in the moderately constricted CHMP1B-only filaments.

Left, central slice along the helical axis of the membrane-bound CHMP1B-only tubule. Right, zoomed view of boxed area in left showing very little dimpling in the outer leaflet (black dashed curved line) of the bilayer. A CHMP1B helix α1, which sits against the membrane, is highlighted in dark green.

Extended Data Fig. 9 SAXS analysis of liposomes and calculation of bilayer thickness.

a, The small angle scattering intensities for protein-free unilamellar vesicles used in this study. The black line represents the fit to the model. The blue data points were used for fitting. b, Fit results for the liposomes and the resulting thickness, D (Å). The bilayer center, ε2, was fixed at 0, and the magnitude of the central peak, ρ2, was fixed at -1. Data are mean ± s.d.

Supplementary information

Reporting Summary

Supplementary Video 1

Elbow flexing of one CHMP1B subunit.

Supplementary Video 2

Flexing of a full turn of CHMP1B subunits from low to high constriction.

Supplementary Video 3

Real-time recording of membrane tube elongation by CHMP1B and IST1.

Supplementary Video 4

IST1 alone does not promote membrane tube elongation.

Supplementary Video 5

Swinging of two IST1 subunits from initial binding to the CHMP1B filament to the constricted state (side view).

Supplementary Video 6

Swinging of two IST1 subunits from initial binding to the CHMP1B filament to the constricted state (top-down view). CHMP1B and the membrane would lie at the top of the animation.

Supplementary Video 7

Constriction of a membrane tubule by CHMP1B and IST1.

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Nguyen, H.C., Talledge, N., McCullough, J. et al. Membrane constriction and thinning by sequential ESCRT-III polymerization. Nat Struct Mol Biol 27, 392–399 (2020).

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