Tom1 Modulates the Endosomal Function of Tollip via a Folding-Upon-Binding Mechanism

Abstract

Many cellular signaling processes require internalization of ubiquitylated cargo via endocytosis and early endosomes are the first sorting station for vesicular cargo. Adaptor protein complexes modulate signaling output in early endosomes by triggering cargo sorting for lysosomal degradation. Tollip, through its C2 domain, associates with endosomal membranes via phosphatidylinositol 3-phosphate (PtdIns(3)P) and recruits ubiquitylated cargo to these compartments via its C2 and CUE domains. Interestingly, binding of Tollip to PtdIns(3)P is inhibited by ubiquitin. Tom1, through its GAT domain, is recruited to endosomes by binding to cargo and the Tollip TBD region by an unknown mechanism. NMR data revealed that Tollip TBD is a natively unfolded domain that partially folds when bound to Tom1 GAT. Furthermore, the association of Tom1 to Tollip inhibits Tollip's binding to PtdIns(3)P. We hypothesize that Tom1 GAT binding to Tollip TBD causes conformational changes in Tollip that impairs its binding to PtdIns(3)P, increasing its commitment to protein sorting. Tollip TBD-Tom1 GAT forms a stable heterodimer, whose association is mainly driven by hydrophobic contacts of high affinity. Nuclear spin relaxation studies demonstrate that the N-terminal half structure of Tollip TBD, which contacts Tom1 GAT, is ordered, whereas the C-terminal half is highly unstructured. Ubiquitin binds to Tom1 GAT at a site that does not overlap with that for the Tollip TBD following a fast exchange regime. Cytosolic Tom1 is recruited to endosomal compartments when co-expressed with Tollip in mammalian cells, but mutations, which reduce 90,000-fold the association of these proteins, revert this effect. Accordingly, we propose that association of Tom1 to Tollip facilitate Tollip release from endosomal membranes, allowing Tollip to commit to endosomal cargo trafficking.I respectfully request the Committee consider our work for oral presentation based on its interest to a broad audience.