Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.

Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.