Role of Modular Proteins in Endosomal Protein Trafficking

Ubiquitylation is a highly controlled post-translational modification of proteins, in which proteins are conjugated either with monoubiquitin or polyubiquitin chains. Ubiquitin modifications on target proteins are recognized by ubiquitin-binding domains, which are found in several effector proteins. In this study, we describe the function of the Toll-interacting protein (Tollip), which is an effector protein in the innate immune signaling pathway and an adaptor protein for endosomal trafficking. We have previously demonstrated that the central C2 domain of Tollip preferentially interacts with phosphoinositides and that this association is critical for membrane targeting of the protein. Remarkably, we found that ubiquitin modulates Tollip’s lipid binding. We have observed an ubiquitin dose-dependent inhibition of binding of Tollip to phosphoinositides and it does so specifically by blocking Tollip C2 domain-phosphoinositide interactions. This led us to hypothesize that the Tollip C2 domain is a novel ubiquitin-binding domain. In addition, we have biophysically characterized the association of the Tollip CUE domain to ubiquitin and identified key interacting residues. The Tollip CUE domain reversibly binds ubiquitin with low micromolar affinity at a site that overlaps with that corresponding to the Tollip C2 domain. We have also found that ubiquitin binding to dimeric Tollip CUE domain induces a drastic conformational change in the protein, leading to the formation of a heterodimeric Tollip CUE-ubiquitin complex. These data suggest that ubiquitin binding to the Tollip C2 and CUE domain and ubiquitin-mediated dissociation of CUE dimer reduces the affinity of the Tollip protein for endosomal phosphoinositides, allowing Tollip cytoplasmic sequestration. Overall, our findings will provide the structural and molecular basis to understand how Tollip, as an endosomal adaptor protein, is modulated by ubiquitin and determines the fate of polyubiquitinated cargo for endosomal degradation. This project is in collaboration with Carla Finkielstein (Biological Sciences, Virginia Tech) and Chenggang Tao (Physics, Virginia Tech).

 

Daniel G. S. Capelluto
Associate Professor
Biological Sciences Department
Biocomplexity Institute 263C
1015 Life Science Circle
Blacksburg, VA 24061-0477
E-mail: capellut@vt.edu
Phone: (540) 231-0974

College of Science

 

 

Department of Biological Sciences
2125 Derring Hall
Mail Code 0406
Virginia Tech
Blacksburg, VA 24061-0406
Phone: (540) 231-8930
Fax: (540) 231-9307

Last update 31 May 2017