Increased mitochondrial leucyl- and phenylalanyl-tRNA synthetase activity as a result of inhibition of mitochondrial protein synthesis.

Abstract

THE synthesis of certain mitochondrial proteins is inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis, and not by the inhibitors of mitochondrial protein synthesis, chloramphenicol and ethidium bromide1,2. This suggests that these proteins are synthesised on cytoplasmic ribosomes from messenger RNA (mRNA) transcribed in the nucleus. Their formation, however, is dependent, in some way, on mitochondrial protein synthesis, as growth in the presence of inhibiting concentrations of either ethidium bromide or chloramphenicol results in great increases in the specific activity of mitochondrial DNA-dependent RNA polymerase1, elongation factors, and methionyl-tRNA transformylase, as well as an increase in mitochondrial ribosomal proteins2. This led Barath and Kuntzel1 to propose that the increased synthesis of mitochondrial proteins results from a disruption of mitochondrial control over transcription of nuclear genes specifying the structure of mitochondrial proteins. The model proposes a represser protein, specified and produced by the mitochondrion, that regulates transcription of mitochondrion-specific genes in the nucleus. When synthesis of the putative represser is inhibited by either ethidium bromide or chloramphenicol, the target nuclear genes are transcribed at an increased rate and the production of mitochondrial proteins increases. Although a direct test of Barath and Kuntzel's model would involve identifying the repressor and its targets, some of the underlying assumptions may be checked by measuring the production by Neurospora of the mitochondrial leucyl-tRNA synthetase, whose structure is known to be determined by a nuclear gene in the Leu-5 region of linkage group V (refs 3 and 4, and our unpublished results). In this case the site of mRNA synthesis is deduced from formal genetic analysis rather than from patterns of inhibition by inhibitors of protein synthesis. As an internal control we also followed the synthesis of the mitochondrial phenylalanyl-tRNA synthetase (whose genetics is unknown). We found that the synthesis of the leucyl-tRNA synthetase in response to chloramphenicol, ethidium bromide and cycloheximide fully conformed to Barath and Kuntzel's model. The production of phenylalanyl-tRNA synthetase activity in response to the inhibitors, however, suggests the involvement of either activation of extant enzyme or processing of an inactive precursor molecule as a large component of the increase in the specific activity of the enzyme after treatment with chloramphenicol.