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Outer and inner membranes of Neurospora crassa mitochondria were separated by the combined swelling, shrinking, sonication procedure. Membranes were characterized by electron microscopy and by marker enzyme activities. A red carotenoid pigment was found to be concentrated in the outer membrane. The inner mitochondrial membrane was resolved into about 20 protein bands on polyacrylamide gel electrophoresis, whereas the outer membrane shows essentially one single protein band. Only negligible incorporation of radioactive amino acids occurs into outer membrane when isolated mitochondria are synthesizing polypeptide chains. In agreement with this observation labeling of outer membrane protein is almost entirely blocked, when whole Neurospora cells are incubated with radioactive amino acids in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Finally, the essential electrophoretic protein band from outer membrane does not become labeled when mitochondria are incubated with radioactive amino acids either in vitro or in vivo in the presence of cycloheximide. It is concluded that the vast majority, if not all, of the outer membrane protein is synthesized by the cytoplasmic system and that polypeptide chains formed by the mitochondrial ribosomes are integrated into the inner mitochondrial membrane.