Large-scale purification of mitochondrial protein complexes in yeast expression system for structural analyses

Recent developments in cryo-electron microscopy techniques have facilitated intensive research into determining protein structures. Nevertheless, the structures of some mitochondrial membrane protein complexes remain undetermined. One possible reason for this research gap is that mitochondrial membrane protein complexes are difficult to overexpress and purify. Even using high-resolution cryo-electron microscopy, structural determination is not possible without first obtaining purified homogeneous proteins. As determining novel structures of protein complexes would provide opportunities to answer many unresolved biological questions, it is important to generalize purification methods, which often become bottlenecks in protein research. In this chapter, we introduce purification methods for mitochondrial membrane protein complexes and mitochondria-localized soluble protein complexes using a yeast expression system. We also describe the recent development of a mitochondrial membrane isolation method that enables the extraction of large amounts of protein complexes for structural analyses.