title: Ubiquitin interacts with the Tollip C2 and CUE domains and inhibits binding of Tollip to phosphoinositides.
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.
authors.affiliation: From the Department of Biological Sciences, Protein Signaling Domains Laboratory, Virginia Tech, Blacksburg, Virginia 24061.
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Mitra
authors.affiliation: From the Department of Biological Sciences, Protein Signaling Domains Laboratory, Virginia Tech, Blacksburg, Virginia 24061.
authors.forename: C Alicia
authors.initials: CA
authors.lastname: Traughber
authors.affiliation: From the Department of Biological Sciences, Protein Signaling Domains Laboratory, Virginia Tech, Blacksburg, Virginia 24061.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Brannon
authors.affiliation: From the Department of Biological Sciences, Protein Signaling Domains Laboratory, Virginia Tech, Blacksburg, Virginia 24061.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Gomez
authors.affiliation: From the Department of Biological Sciences, Protein Signaling Domains Laboratory, Virginia Tech, Blacksburg, Virginia 24061. Electronic address: capellut@vt.edu.
authors.forename: Daniel G S
authors.initials: DGS
authors.lastname: Capelluto
mesh_terms: D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D019906:Nuclear Magnetic Resonance, Biomolecular; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; C410602:TOLLIP protein, human; D025801:Ubiquitin
keywords: Endosomes; Membrane Trafficking; Nuclear Magnetic Resonance; Phosphoinositides; Ubiquitin