Abstract
The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.
Highlights
ESCRT proteins catalyze membrane budding and fission away from the cytosol
CHMP2B Localizes to Plasma Membrane upon VPS4 Knockdown—Immunostaining of CHMP4A, -B, and -2B revealed that all endogenous proteins were distributed homogeneously throughout the cytoplasm. siRNA knockdowns of VPS4A and VPS4B led to a relocalization of cytosolic CHMP4A and CHMP4B to intracellular patches, possibly reflecting their accumulation at the surface of endosomes (Fig. 1, A and B)
Similar depletion of VPS4 led to relocalization of endogenous CHMP2B from the cytoplasm into patches, which were associated with the plasma membrane in some cells (Fig. 1, C and D, and supplemental Fig. S2B)
Summary
Results: The ESCRT-III protein CHMP2B polymerizes into tubular helical structures deforming the plasma membrane. CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission This model awaits the demonstration that such structures exist and can deform membranes in cellulo. It has been hypothesized that the tight interaction of the dome with the membrane at the neck of the budding vesicle might lead to the closing of the neck and spontaneous membrane fission [33] In contrast to these structures observed in vitro, CHMP4B overexpression in cultured cells led to its assembly into circular filaments at the plasma membrane. Cryo-electron microscopy of membrane tubes shed into the cell culture supernatant demonstrates that CHMP2B polymerizes into a tightly packed helical polymer intimately associated with the inner leaflet of the bilayer. Our data demonstrate that in mammalian cells, CHMP2B is an adaptor protein for the recruitment of VPS4 as demonstrated in yeast and directly plays a structural role in membrane scaffolding
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