Identification of NPC1-interacting Proteins at ER-Lysosome Contact Sites via TurboID-mediated Proximity Labeling and In silico Analysis

Abstract

Cholesterol is essential for all eukaryotic cells and is involved in a variety of cellular processes. Although cholesterol is synthesized within the cell, large portions of intracellular cholesterols are obtained through receptor-mediated endocytosis. Endocytosed cholesterols are transported to the lysosome and then distributed to various membrane compartments from the lysosome. This distribution requires Niemann-Pick type C1 (NPC1), a lysosomal membrane protein. It has been suggested that NPC1 transports cholesterols through a membrane contact site (MCS) between two distinct organelles. However, proteins that interact with NPC1 for cholesterol trafficking through a MCS remain poorly understood. In this study, therefore, I aim to discover proteins involved in cholesterol transport via an ER-lysosome MCS using TurboID-mediated proximity labeling and in silico analysis. TurboID utilizes biotin and ATP to covalently biotinylate neighboring proteins, allowing for the identification and characterization of the dynamic network involved in intricate molecular interplays. By analyzing proteins biotin-labeled by NPC1-TurboID using mass spectrometry, candidates for ER-resident proteins interacting with NPC1 possibly at ER-lysosome MCS will be isolated. GO analyses for these proteins showed significant associations with cholesterol homeostasis and endoplasmic reticulum for the biological process and the cellular component, respectively.