Structural and Functional Basis of Alternative ESCRT-0 Protein Complexes

Abstract

Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Cargo transport is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. Similar to the structural organization of ESCRT-0 proteins, alternative ESCRT-0 (alt-ESCRT-0) proteins, such as Tollip and Tom1, also present multiple ubiquitin-binding domains, including the C2 and CUE (Tollip) and VHS and GAT (Tom1) domains. Tollip localizes the Tollip-Tom1 complex at endosomal compartments by association with phosphatidylinositol 3-phosphate (PtdIns(3)P) through its central C2 domain. Tom1, through its GAT domain, is recruited to endosomes by binding to Tollip's Tom1-binding domain (TBD) through an unknown mechanism. Our NMR data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N-terminus when bound to the first two helices of the Tom1 GAT domain through high affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Binding of the Tollip C2 domain is mediated by the third helix of the Tom1 GAT domain. We also show that the Tom1 GAT domain is able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors Tollip's release from endosomal membranes, allowing Tollip to commit to cargo trafficking. More recently, Tom1 has been shown to associate to signaling endosomal PtdIns(5)P through its VHS domain delaying epidermal growth factor receptor degradation in a bacterial infection model. We found that PtdIns(5)P binding of the Tom1 VHS domain is in the moderately fast-exchange regime at the NMR timescale. We propose that an incremental change in PtdIns(5)P intracellular levels sequesters Tom1 in endosomal cargo-free membrane domains, keeping the protein from its cargo sorting function.