Mapping the Emerin Interactome by Apex Proximity Labeling

Abstract

Emerin is a highly conserved LEM-domain protein expressed mostly at the inner nuclear envelope of mammalian cells. Mutations in the EMD gene encoding for emerin cause X-linked Emery-Dreifuss muscular dystrophy (EDMD). Through its interaction with a variety of nuclear binding partners, emerin has a number of biological functions that include regulation of gene expression, nucleo-cytoskeleton mechanotransduction and maintenance of nuclear shape. The molecular pathogenesis of EDMD remains unclear and mapping the protein interaction network of emerin can potentially help elucidate the molecular mechanisms behind the disease. Here we used a proximity-dependent biotinylation method based on an engineered ascorbate peroxidase (APEX2) to obtain proteomic profiles of emerin interactions at the nuclear envelope and the endoplasmic reticulum in cells. We generated constructs of APEX2 fused to the N- or the C-terminus of emerin to study its interacting partners across the inner nuclear envelope, the outer nuclear envelope and the endoplasmic reticulum membrane. We also optimized proximity biotin labeling in both live and fixed cells, and showed that biotinylation patterns vary significantly between emerin fusions and for cells micropatterned on fibronectin strips of decreasing widths that we use to apply increasing mechanical perturbation to the cell nuclei. These data indicate that emerin explores different microenvironments and interacts with distinct partners as a function of nuclear strain. Using APEX2 quantitative proteomics, we also started to identify key interacting partners of emerin and their subcompartmental localizations for cells subjected to increasing mechanical challenges.