Protein crowding mediates membrane remodeling in upstream ESCRT-induced formation of intraluminal vesicles

Susanne Liese,Eva Maria Wenzel,Ingrid Kjos,Rossana Rojas Molina,Sebastian W Schultz,Andreas Brech,Harald Stenmark,Camilla Raiborg,Andreas Carlson

doi:10.1073/pnas.2014228117

Abstract

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III-depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.

Highlights

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs)
Once the endosomal sorting complex required for transport (ESCRT)-0 signal reaches a maximum in fluorescence intensity, ESCRT-III exhibits a jump in its fluorescence intensity over just a few seconds, before it decreases with a decay time similar to that of ESCRT-0
By combining mathematical modeling and cell biological data we are able to point to the biophysical determinants that facilitate ILV budding by upstream ESCRTs, which is a consequence of the interplay between ESCRT-induced Gaussian bending rigidity and their crowding on the membrane

Summary

Introduction

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). While ESCRT-0, -I, and -II sequester transmembrane cargo proteins and facilitate ILV formation, ESCRT-III and the ATPase VPS4 enable constriction of the membrane neck leading to the formation of an ILV [19]. The only energy-consuming step in the membrane remodeling process is the membrane scission, involving the ATPase VPS4 [20, 21] This is especially remarkable, since the energy required for a flat lipid bilayer to form a spherical vesicle in absence of a protein coat is orders of magnitude larger than the thermal energy, creating an energy barrier inhibiting vesicle formation [22, 23]. Transmission electron microscopy (TEM) images and electron tomography have provided high-resolution

Methods

Results

Conclusion