Posttranslational mitochondrial protein import in a homologous yeast in vitro system

Posttranslational import of preproteins into mitochondria has been reported to be inefficient in a homologous yeast in vitro system, suggesting a requirement for coupling of protein synthesis and import. We have characterized a homologous yeast in vitro system which allows posttranslational mitochondrial import of preproteins. The efficiency is comparable to that of the heterologous system with rabbit reticulocyte lysate and isolated yeast mitochondria. Import in the homologous system depends on mitochondrial surface receptors, a membrane potential and the matrix heat shock protein Hsp70. Import is not blocked by the protein synthesis inhibitor cycloheximide, but is impaired by induction of stable folding in preproteins. Our studies demonstrate a posttranslational translocation mechanism in the homologous system, strongly supporting the validity of conclusions drawn from the widely used heterologous import system.