Directing proteins to mitochondria by fusion to mitochondrial targeting signals

Specific targeting mechanisms have been identified for the various subcellular compartments in eukaryotic cells. These processes are mediated by dedicated translocation machineries. Most mitochondrial proteins are synthesized in the cytosol as larger precursors carrying a positively charged, N-terminal presequence that is cleaved by a specific protease on import into the innermost compartment, the mitochondrial matrix. In this pathway, the precursor is bound by cytosolic chaperones, and then delivered to a set of receptors on the outer surface of the organelle. Many of the features of protein import into mitochondria, both in terms of identification of the components involved and the molecular mechanisms, has been dissected by using hybrid proteins. Almost every protein when fused to a mitochondrial matrix-targeting signal can be directed to the mitochondrial matrix. The chapter describes the use of hybrid proteins for in vitro targeting to isolated mitochondria from the yeast Saccharomyces cerevisiae.