Monitoring alpha-helical membrane protein insertion into the outer mitochondrial membrane in mammalian cells

Mitochondrial function is dependent on the correct localization and insertion of membrane proteins into the outer mitochondrial membrane (OM). In mammals, the OM contains ∼150 proteins, the majority of which contain α-helical transmembrane domains. This family of α-helical proteins has significantly expanded in metazoans and has evolved to mediate critical signaling and regulatory processes including mitochondrial fusion and fission, mitophagy, apoptosis and aspects of the innate immune response. Recently, the conserved OM protein MTCH2 has been identified as an insertase for α-helical proteins in human mitochondria. However, our understanding of the targeting, insertion, folding and quality control of α-helical OM proteins remains incomplete. Here we highlight three methods to monitor α-helical protein insertion both in human cells and in vitro. First, we describe a versatile split fluorescent reporter system that can be used to monitor the insertion of α-helical proteins into the OM in human cells. Second, we delineate a streamlined approach to isolating functional, insertion competent mitochondria from human cells that are compatible with in vitro import assays. Finally, we explain in detail how to reconstitute the insertion of α-helical proteins in a minimal system, by creating functional proteoliposomes containing purified MTCH2. Together these tools represent an integrated platform to enable the detailed mechanistic analysis of biogenesis of the diverse and physiologically essential α-helical OM proteome.