Identification of Atlastin-interacting Proteins Using TurboID-mediated Proximity Labeling

Abstract

The endoplasmic reticulum (ER) plays various roles in the cell, such as protein and lipid synthesis, protein translocation, and calcium homeostasis. These functions are believed to be closely related to the network structure of the ER, but detailed molecular mechanisms are still uncovered. The formation and maintenance of the ER network is regulated by homotypic ER membrane fusion, and this ER membrane fusion is mediated by atlastin proteins. In order to study their functions, identification of novel atlastin-interacting proteins is essential. In this study, I identified potential atlastin-binding proteins by TurboID-mediated proximity labeling. TurboID is a mutant E. Coli biotin ligase (BirA) that uses biotin and ATP to covalently attach biotin to the lysine residues of adjacent proteins. Due to its short working radius, it has been widely used to identify protein complexes which have transient or weak interactions between their components. Using this technique, I generated a DNA construct encoding a human atlastin-2 protein fused with TurboID and introduced it in HeLa cells. Mass spectrometric analysis of biotinylated proteins identified Sec22b protein as a strong candidate for an atlastin-2 interacting protein. In future studies, I will examine whether Sec22b physically interacts with atlastin-2 by co-immunoprecipitation, and investigate how Sec22b might co-operate with atlastin-2 to regulate ER membrane fusion.