APEX, a Master Key To Resolve Membrane Topology in Live Cells.

Determining the topology of the membrane proteome is fundamental for understanding its function at the membrane. However, conventional methods involving test tube reactions often lead to unreliable results, which do not accurately reflect membrane topology under physiological conditions, as perturbations occur during lysis. In this Perspective, we introduce a new method using engineered ascorbate peroxidase (APEX) for revealing membrane topological information in live cells without performing complicated sample preparation. We also discuss several examples of clearly resolved membrane topologies of various important mitochondrial proteins (e.g., LETM1, NDUFB10, MCU, SFXN1, and EXD2) and endoplasmic reticulum proteins (e.g., HMOX1) determined by using APEX-based methods.