Proximity interactions of the ubiquitin ligase Mind bomb 1 reveal a role in regulation of epithelial polarity complex proteins

Sascha E Dho,Nancy Silva-Gagliardi,C Jane Mcglade,Brian Raught,Etienne Coyaud,Fabio Morgese,Emily Lamoureux,Donna M Berry

doi:10.1038/s41598-019-48902-x

Abstract

MIB1 belongs to the RING domain containing family of E3 ubiquitin ligases. In vertebrates, MIB1 plays an essential role in activation of Notch signaling during development, through the ubiquitination and endocytosis of Notch ligands. More recently, Notch independent functions for MIB1 have been described in centriole homeostasis, dendritic spine outgrowth and directional cell migration. Here we use proximity-dependent biotin identification (BioID) to define the MIB1 interactome that included 163 high confidence interactions with polypeptides linked to centrosomes and cilia, endosomal trafficking, RNA and DNA processing, the ubiquitin system, and cell adhesion. Biochemical analysis identified several proteins within these groups including CCDC14 and EPS15 that were ubiquitinated but not degraded when co-expressed with MIB1. The MIB1 interactome included the epithelial cell polarity protein, EPB41L5. MIB1 binds to and ubiquitinates EPB41L5 resulting in its degradation. Furthermore, MIB1 ubiquitinates the EPB41L5-associated polarity protein CRB1, an important determinant of the apical membrane. In polarized cells, MIB1 localized to the lateral membrane with EPB41L5 and to the tight junction with CRB1, CRB3 and ZO1. Furthermore, over expression of MIB1 resulted in altered epithelial cell morphology and apical membrane expansion. These results support a role for MIB1 in regulation of polarized epithelial cell morphology.

Highlights

MIB1 belongs to the RING domain-containing family of E3 ligases
To identify the human MIB1 proximity interactome, a BirA (R118G) biotin ligase-MIB1 fusion protein was expressed in HEK293 cells in the presence of excess biotin and purified biotinylated protein species identified by liquid chromatography (LC)-MS/MS (Supplementary Table S1)
During www.nature.com/scientificreports the course of this study, similar co-localization data were reported by Matsuda et al.[39] and our findings extend these observations by showing that relocalization of MIB1 by EPB41L5 occurs in a manner that does not depend on MIB1 E3 ligase activity

Summary

Introduction

MIB1 belongs to the RING domain-containing family of E3 ligases. It is a multi-domain protein that includes an amino terminal MZM region composed of two regions of homology to the HERC2 ubiquitin ligase (MIB/HERC domains) flanking a ZZ type zinc finger, two adjacent MIB homology repeats (REP), and a central region containing Ankyrin repeats[1,2,3]. MIB1 has a conserved function in the activation of Notch signaling during development through the ubiquitination and endocytosis of Notch ligands[2,6,7,8]. In Drosophila, both MIB1 and another structurally distinct RING domain E3, Neuralized, play overlapping and discrete roles in the regulation of Notch ligand endocytosis while in vertebrates, only MIB1 appears to be required for Notch signalling[8]. In addition to the conserved and well-defined role in the regulation of Notch pathway activation, the identification of functions unrelated to Notch pathway activation reveal that in mammalian cells MIB1 regulates a broad range of cellular functions. We identify interactions with the polarity proteins EPB41L5 and Crumbs and describe a novel role for MIB1 in regulating in the maintenance of epithelial cell shape and morphology

Methods

Results

Conclusion