Nuclear protein synthesis in a temperature-sensitive Chinese hamster ovary cell line at 40 degrees C.
We examined the incorporation of radioactive amino acids into nuclear proteins occurring at nonpermissive conditions in tsH1 Chinese hamster ovary cells with a temperature-sensitive defect in cytosol nonmitochondrial protein synthesis. In leucine-free medium at 40 degrees C, total cellular protein synthesis declined by 1-1.5%/min. As reported by others, preincubating these cells at 42 degrees C for 5-10 min sharply increased the rate of decline. The synthesis of acidic nuclear proteins at nonpermissive conditions (40 degrees C + 300 micrograms/ml cycloheximide) was demonstrated by the nuclear incorporation of 3H-tryptophan. Radioactivity, seen by autoradiography to be associated with these isolated Triton-X-100-washed nuclei, was released after incubating labelled nuclei with proteolytic enzymes. During incubation of tsH1 cells at nonpermissive conditions, pulse/chase experiments were consistent with the loss of some nuclear radioactivity into the cytoplasm. The distribution of cytosol and nuclear proteins, labelled at permissive or nonpermissive conditions and separated by isoelectric focusing, differed quantitatively and probably qualitatively, confirming the residual synthesis of acidic nuclear proteins at 40 degrees C in the presence of cycloheximide. Most newly synthesized acidic proteins retained by nuclei from cells labeled at nonpermissive conditions were present in a transciptionally active chromatin fraction. Although under these conditions the apparent rate of cellular RNA synthesis was unchanged, inhibiting residual cycloheximide-resistant nuclear protein synthesis with puromycin proportionately reduced RNA synthesis. Preincubating cells with 20 micrograms/ml of actinomycin D did not inhibit residual labelling of nuclear proteins; effects on residual nuclear labelling of impaired mitochondrial respiration were ambiguous. Nuclear proteins labelled under nonpermissive conditions probably included some of the 'prompt' heat shock proteins recently described. Provided certain assumptions are correct, our results are consistent with very limited protein synthesis associated with and even intrinsic to cell nuclei. They also suggest that this residual cycloheximide-resistant protein synthesis could be concerned with optimum synthesis or processing of certain nuclear RNA species.
D000818:Animals; D002460:Cell Line; D002701:Chloramphenicol / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003513:Cycloheximide / Q000494:pharmacology; D005260:Female; D007930:Leucine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D009698:Nucleoproteins / Q000096:biosynthesis*; D010053:Ovary / Q000187:drug effects / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism
D009698:Nucleoproteins; D011691:Puromycin; D012313:RNA; D002701:Chloramphenicol; D014364:Tryptophan; D003513:Cycloheximide; D008715:Methionine; D007930:Leucine