In vitro translation in yeast extract to study interactions with cytosolic chaperones

Saccharomyces cerevisiae, a well-established model organism, serves as a valuable tool for unraveling various eukaryotic cellular processes. Its utility extends to studying protein folding and interactions with chaperones, as it demonstrates a capability to produce and process proteins in a manner closely resembling that of higher eukaryotes. To gain insights into the events taking place following protein synthesis in the cytosol, an in vitro translation system is essential-one that mirrors in vivo processes yet allows for easy manipulation. To address this need, multiple protocols for preparation of cell-free translation lysates from S. cerevisiae have been documented. This chapter introduces an optimized and modified in vitro pull-down approach following protein translation in yeast cell-free extract. The advantages of this system in investigating the interactions of newly synthesized mitochondrial proteins with cytosolic chaperones are described. This procedure exploits the dual advantages of yeast cell-free lysates, serving as both a protein synthesis tool, and a reservoir for cytosolic factors and chaperones. In summary, the depicted approach provides a versatile platform that deepens our understanding of the early cytosolic events in the biogenesis of nascent proteins before reaching their ultimate organelle.