The Biosynthesis of Rat-Liver Cytochrome c. 2. Subcellular Distribution of Newly Synthesized Cytochrome c

Early events in the biosynthesis of rat liver cytochrome c were investigated on animals killed at times ranging between 5 min and 2 h after receiving an intravenous injection of l-[3H]leucine. Subcellular fractions were separated by various procedures, including differential sedimentation and density equilibration in a sucrose gradient. The radioactivity associated with cytochrome c was measured by improved techniques which involved partial purification by means of ion-exchange chromatography, followed by immunoprecipitation by a double-antibody technique. It was found that label appears in mitochondrial cytochrome c as early as 5 min after injection of the labeled amino acid, with no measurable lag and with no indication of the existence of an extramitochondrial precursor pool. In confirmation of the observations of other workers, microsomal cytochrome c was found to have an apparent specific radioactivity higher (by a factor of 2) than that of mitochondrial cytochrome c. However, various experiments as well as kinetic considerations have ruled out a precursor role for microsomal cytochrome c; it seems very likely that traces of a highly labeled contaminant are responsible for the excess radioactivity recovered with microsomal cytochrome c. It is concluded that cytochrome c is either synthesized in some unidentified extra-mitochondrial site and transferred immediately to the mitochondria, or synthesized and transferred to the mitochondria simultaneously by cytoplasmic ribosomes attached to the outer mitochondrial membrane.