Biogenesis of Cytochrome c in Neurospora crassa. Synthesis of Apocytochrome c, Transfer to Mitochondria and Conversion to Holocytochrome c

Abstract

1 Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2 Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3 Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labelling kinetics do not suggest a precursor role of microsomal apocytochrome c or holocytochrome c. 4 Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5 Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6 The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The probles of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.