Structural and Functional Basis of Tollip Association to the Endosomal Adaptor Protein Tom1

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Adaptor proteins are often committed to cellular processes that involve cargo internalization from the plasma membrane. Ubiquitinated cargo is internalized by endocytosis and delivered to early endosomes via intracellular vesicles. Cargo is then sorted to late endosomes/multivesicular bodies followed by, in most cases, degradation in the lysosomal compartments. Adaptor proteins, such as Tollip and Tom1, facilitate cargo sorting through their ubiquitin-binding domains. Tollip is localized to early endosomes, through binding to phosphatidylinositol 3-phosphate (PtdIns(3)P). Tom1 can also bind ubiquitin-conjugated cargo and is recruited to the endosomal membranes through its association with Tollip. The interaction of these two proteins is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. In this work, we demonstrate that binding of Tollip to PtdIns(3)P is negatively modulated by interaction with Tom1. Structural studies determine that the Tom1-binding domain (TBD) of Tollip is intrinsically disorded and folds upon binding to the Tom1 GAT domain, which also undergoes a conformational change upon binding. Intermolecular NOEs of the Tollip TBD-Tom1 GAT complex indicate that association is mainly driven by hydrophobic contacts with very high affinity. Ubiquitin binds to the Tom1 GAT domain at a site that does not overlap with that for the Tollip TBD, but the binding events are mutually exclusive and are likely driven by conformational changes in the GAT domain. Endosomal localization of Tom1 depends on the presence of Tollip in this compartment. Using fluorescence microscopy, we show that mutations in the binding interphase of the Tom1 GAT and Tollip TBD complex leads to a dissociation of the proteins and triggers cytosolic localization of Tom1. Consequently, we propose that association of Tom1 to Tollip helps to release Tollip from endosomal membranes, allowing Tollip to commit to endosomal ubiquitinated cargo trafficking.