In vitro analysis of yeast mitochondrial protein import.

This unit describes methods for importing in vitro-translated or recombinant proteins into isolated yeast mitochondria and for exporting mitochondrial proteins translated in the yeast mitochondrial matrix into the inner mitochondrial membrane. The methods use mitochondria isolated from yeast cells and mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate mitochondrial matrix translation of proteins and their export.