Endosomal sorting complex required for transport (ESCRT) complexes induce phase-separated microdomains in supported lipid bilayers

Abstract

The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.

FIGURE 1.

ESCRT-II clusters on SLBs. A, 10 nm human ESCRT-II was loaded into the flow cell, and formation of clusters was observed. B, histogram of human ESCRT-II cluster sizes obtained from quantification of 100 clusters and divided into 20 molecule bins. C, PI(3)P binding FYVE domain does not form clusters on the SLB. 50 nm GFP-EEA1-FYVE domain was loaded into the flow cell for this experiment. D, yeast ESCRT-II forms clusters on SLBs under the same conditions as in A, but the clusters are smaller and more numerous than for human ESCRT-II. (E). Human ESCRT-II forms well resolved discrete clusters on the PI(3)P-containing GUVs. Scale bar, 10 μm.

FIGURE 2.

Stoichiometric binding of membrane-tethered ubiquitin. A–C, 100 nm Alexa488-labeled His₆-tagged ubiquitin (A) and 20 nm Atto647-labeled ESCRT-II (B) were loaded together into the flow cell. Formation of ESCRT-II clusters that co-localized with ubiquitin was observed, and the stoichiometry was estimated from the intensity of fluorescence. D--F, ubiquitin-I44D does not co-localize with ESCRT-II. Scale bar, 10 μm.

FIGURE 3.

Stoichiometric assembly of VPS20 with ESCRT-II clusters. A–C, VPS20 and ESCRT-II were both loaded at 5 nm concentration into the flow cell, and co-localization was observed (upper panel). D--F, mutant VPS20^ΔESCRT-II that is crippled for ESCRT-II binding in solution did not form clusters with ESCRT-II even at 30 nm. Scale bar, 10 μm.

FIGURE 4.

ESCRT-II and VPS20 co-assemble over time on the molecular scale. A, 10 nm of Alexa488-labeled ESCRT-II was loaded into the flow cell. Several hundred frames later to be sure that the equilibrium in the flow cell was reached, 20 nm rhodamine-labeled VPS20 was added. FRET was observed as a specific quench of the donor fluorescence upon arrival of the acceptor. A single cluster is highlighted by an arrow (A), and fluorescence data over time are plotted for this single cluster (B). Scale bar, 10 μm.

FIGURE 5.

ESCRT-II clustering is cholesterol-dependent. ESCRT-II was injected on membranes where cholesterol was replaced by POPC in the standard 25% cholesterol mixture to yield the indicated mole fractions of cholesterol. We observed no clustering at low cholesterol concentrations (A and B; 0 or 10%), and more diffuse clustering on the membrane containing 15% cholesterol (C) was compared with discrete clusters seen on the membrane with 25% cholesterol (D). E, dioleoyl PI(3)P supports clustering in combination with 25% cholesterol. Scale bar, 10 μm.

FIGURE 6.

Lipid phase separation is associated with ESCRT clustering. A, 20 nm unlabeled ESCRT-II and 40 nm VPS20-Alexa488 were loaded into a flow cell over an SLB containing 0.1% DiD (B). ESCRT-II clusters exclude the dye. C, merge of A and B. Scale bar, 10 μm.