An assessment of in vivo mitochondrial protein synthesis in Neurospora crassa.

This paper describes a procedure which was designed to maximally inhibit the synthesis in vivo of all proteins except those synthesized in the mitochondria. To this end a 15min preincubation of Neurospora crassa cells with 100 fig of cycloheximide per ml was employed before 14Cleucine was introduced. A small fraction of the protein synthesized by the whole cell (2.15%) was found to be completely resistant to inhibition by cycloheximide and sensitive to inhibition by chloramphenicol. The cycloheximide-resistant, chloramphenicol-sensitive protein-synthesizing system was shown to co-sediment with the mitochondrial fraction in cell homogenates fractionated by sucrose density gradient centrifugation. It was deduced that the mitochondria, in vivo, are the sites of cycloheximide-resistant protein synthesis. The protein they synthesize is equivalent to about 14.5% of the protein of the mitochondrion. Mitochondrial protein synthesis, in vivo, is over 100 times more rapid than the fastest rates yet reported in vitro and is linear for at least 30 min. As such, intramitochondrial protein synthesis in vivo offers the possibility of obtaining proteins of extremely high specific activity and may shed new light on the nature of the protein synthesized by the mitochondria.