Two-dimensional condensates of HRS drive the assembly of flat clathrin lattices on endosomes

Amongst the different clathrin structures in mammalian cells, bi-layered clathrin coat colocalizing with endosomal sorting complex required for transport (ESCRT)-0 remains one of the most ambiguous. Despite being observed for the first time twenty years ago, their structure and how they are assembled remains unknown. Here, we reconstituted in vitro the ESCRT-0 clathrin assembly onto various types of membranes. The ESCRT-0 protein HRS, a known clathrin adaptor on endosomes, was found to form protein condensates. These condensates spread into a thin layer on PI(3)P-rich membranes. Platinum replica electron microscopy revealed that, surprisingly, the assembly of clathrin was different depending on the HRS phase. Protein droplets recruited clathrin as a dense, curved lattice, with many cage-like structures. On two-dimensional condensates, HRS recruited clathrin as a dense flat assembly. Two-dimensional HRS-clathrin condensates promoted the clustering of cholesterol in the underlying membrane, while cholesterol enhanced PI(3)P- dependent HRS recruitment on the membrane. On free-standing membranes, two-dimensional HRS-clathrin condensates promoted membrane flattening. Overall, these results show that a two- dimensional HRS condensate creates a unique membrane structure for sorting cargo molecules, defining a new mechanism in membrane trafficking processes.