Expression of the mitochondrial genome in HeLa cells: XIII. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis

Abstract

The effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis by cycloheximide or chloramphenicol, respectively, upon mitochondrial DNA and RNA synthesis in HeLa cells was investigated. Cells were treated with cycloheximide or chloramphenicol, and then pulsed with either a DNA or RNA precursor in the presence of the drug. The specific activities of the appropriate nucleoside triphosphate pools were determined to convert labeling rates into synthesis rates. Chloramphenicol-treated cells continue to grow with a normal 24-hour generation time for two generations, and show an additional half generation of growth at a reduced rate, entering a stationary phase after three days of growth. Synthesis of mitochondrial DNA and RNA continues at a normal or near normal rate, on a per cell basis, in HeLa cells grown in the presence of chloramphenicol for three days. The sedimentation profile of pulse-labeled mitochondrial DNA and RNA extracted from chloramphenicol-treated cells is normal, except for a reduced labeling of 4 s RNA after chloramphenicol treatment for three days. The rate of total cell DNA and RNA synthesis is normal or near normal after chloramphenicol treatment. Mitochondrial malate dehydrogenase activity per cell remains constant, or nearly so, during two-day growth in the presence of chloramphenicol, and increases by 30 to 60% in the third day, whereas cytochrome c oxidase activity per cell is diluted proportionally to growth. Synthesis of mitochondrial DNA and RNA is depressed gradually during a four-hour treatment of HeLa cells with cycloheximide, up to a 55 and 80% inhibition, respectively. Under the same conditions, total cell RNA synthesis is also progressively depressed, up to about 60% inhibition, whereas total cell DNA synthesis is inhibited almost completely already after 15 minutes of cycloheximide treatment. The synthesis of all sedimentation classes of mitochondrial RNA appears to be equally affected by cycloheximide treatment in vivo. Synthesis of presumptive oligomeric forms of mitochondrial DNA is less affected than that of other forms of mitochondrial DNA by the same treatment.