VHS domains of ESCRT‐0 cooperate in high‐avidity binding to polyubiquitinated cargo

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT‐0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain–ubiquitin complex was solved at 2.6 Å resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS‐domain containing proteins in yeast and humans, including both subunits of ESCRT‐0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63‐linked polyubiquitinated cargo. Intact human ESCRT‐0 binds Lys63‐linked tetraubiquitin 50‐fold more tightly than monoubiquitin, though only 2‐fold more tightly than Lys48‐linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT‐0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin‐binding sites in yeast ESCRT‐0 shows that cooperation between them is required for the sorting of the Lys63‐linked polyubiquitinated cargo Cps1 to the vacuole.