Abstract
A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.
Highlights
Tollip participates in endosomal membrane trafficking and innate immune pathways
Ubiquitin Inhibits the Phosphoinositide Binding of Tollip— Tollip localizes to endosomal membranes and the subcellular localization of the protein is likely associated with its ability to preferentially bind PtdIns(3)P through its central conserved 2 (C2) domain (18)
The results presented in this study demonstrate that phosphoinositide binding of Tollip is negatively modulated by ubiquitin and that this occurs by ubiquitin association to both Tollip C2 and conjugation to endosomal reticulum degradation (CUE) domains
Summary
Tollip participates in endosomal membrane trafficking and innate immune pathways. Results: Ubiquitin inhibits binding of Tollip to phosphatidylinositol 3-phosphate by association to the Tollip C2 and CUE domains and dissociates Tollip CUE domain dimers. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Several ubiquitinbinding domains have been shown to mediate intra-monoubiquitination, leading to a ubiquitin-binding domain-containing protein conformation that exhibits altered function and localization and likely alters its interactions with ubiquitinated cargo during endosomal trafficking (reviewed in Ref. 6). The function of many adaptor proteins is associated with their ubiquitin-binding domains, ensuring the precise sorting of ubiquitinated cargo proteins inside the intralumenal endosomal vesicles. We propose that ubiquitin binds to two independent sites in Tollip, an association that promotes dissociation of the Tollip CUE domain dimers, and direct inhibition of PtdIns(3)P ligation, leading to membrane release of adaptor protein complexes in the absence of polyubiquitinated cargo
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