Confocal microscopy-based radial profile analysis for submitochondrial protein localization

Mitochondria are double-membrane organelles whose functions depend on membrane proteins. Identifying whether proteins localize to the outer mitochondrial membrane (OMM) or inner mitochondrial membrane (IMM) is important but often requires specialized techniques. Here, we developed a confocal laser scanning microscopy (CLSM)-based radial profile analysis for the localization of submitochondrial proteins. Based on this method, cytochrome c oxidase subunit 4 (COX4), adenine nucleotide translocase 2 (ANT2), mitochondrial calcium uniporter (MCU) and calcium-activated potassium channel subunit alpha-1(K_Ca1.1) signals were spatially distinct from translocase of outer mitochondrial membrane 20 (TOM20) signals in isolated mitochondria. Moreover, osmotic shock increased the apparent spatial separation between OMM- and IMM-associated signals, making it easier to distinguish translocase of inner mitochondrial membrane 23 (TIM23) or pore-forming subunit of the mitochondrial ATP-gated potassium channel (MITOK) signals from TOM20 signals in isolated mitochondria. Our approach provides a practical screening tool for assessing submitochondrial protein localization without specialized instrumentation or expertise.