Mechanistic insights into fungal mitochondrial outer membrane protein biogenesis.

The majority of mitochondrial proteins are nuclear-encoded and need to be transported into the mitochondria, including the proteins in the outer mitochondrial membrane. For ß-barrel proteins, the preproteins are initially recognized and imported by the TOM complex, then shuttled to the SAM complex via small Tim proteins. For ⍺-helical proteins, some preproteins are recognized by the TOM complex and imported into the membrane by the MIM complex. In recent years multiple structures of the TOM complex and the SAM complex have been reported, increasing our understanding of the mechanism of protein biogenesis in the outer mitochondrial membrane.

The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease.

The voltage-dependent anion channel (VDAC) is a ß-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson's disease (PD), Friedreich's ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease.

The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.

Cloning and Multi-Subunit Expression of Mitochondrial Membrane Protein Complexes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane environment. The development of the pBEVY vector system has further increased the potential of S. cerevisiae as an expression system by creating a method for expressing multiple proteins simultaneously. This vector system is amenable to the expression and purification of multi-subunit protein complexes. Here we describe the cloning, yeast transformation, and co-expression of multi-subunit outer mitochondrial membrane complexes using the pBEVY vector system.