Protein Crowding Mediates Membrane Remodeling in Upstream ESCRT-induced Formation of Intraluminal Vesicles

Abstract

<jats:title>Abstract</jats:title><jats:p>As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0-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-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 on the membrane induces membrane deformation and facilitates 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.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>Intraluminal vesicle (ILV) formation plays a crucial role in the attenuation of growth factor receptor signaling, which is mediated by the endosomal sorting complex required for transport (ESCRT-0-III/VPS4). The general dogma has been that the upstream ESCRTs (0-II) sequester the receptors at the surface of endosomes and the downstream ESCRTs (III/VPS4) remodel the endosome membrane leading to the abscission and formation of receptor-containing ILVs. We now show that the upstream ESCRTs not only sequester cargo, but in addition play an essential role for the initiation of membrane shape remodeling in ILV budding. Through a combination of mathematical modeling and experimental measurements we show that upstream ESCRTs facilitate ILV budding by crowding with a high density in the membrane neck region.</jats:p></jats:sec>

Diffuso-kinetic membrane budding dynamics

“…The molecular machinery associated with curvature-inducing processes is often complex 7,8 and while some involve active motor proteins, [9][10][11] there is a multitude of proteins that are able to passively induce membrane shape transformations. 8,12 The biophysical mechanisms that induce membrane curvature include the insertion of amphipathic helixes into the bilayer, 13 producing an area difference between the inner and outer membrane leaflet through the binding of large proteins to one membrane side 14,15 or protein crowding. 16,17 Thus, the net effect of any asymmetry between the leaflets of the bilayer due to anchoring inclusions or steric pressure can be represented by the spontaneous curvature.…”