Nuclear protein synthesis at permissive and non-permissive temperatures in a Chinese hamster ovary cell line with a temperature-sensitive defect in cytoplasmic non-mitochondrial protein synthesis

Abstract

Abstract A Chinese hamster ovary cell line with a temperature-sensitive defect in cytoplasmic non-mitochondrial protein synthesis was used to examine protein synthesis thought to be intrinsic to nuclei. Nuclear fractions did not contain whole cells, endoplasmic reticulum or mitochondria, as judged by light and electron microscopy and contaminating microorganisms and PPLO were absent. When cytoplasmic protein synthesis was almost totally suppressed at 40 °C, in some experiments in the presence of cycloheximide, nuclear and mitochondrial proteins continued to be labelled with radioactive leucine for 15–30 min. Nuclear incorporation at 40 °C was suppressed by puromycin and partially inhibited by 225 μg of chloramphenicol per ml. Most of the nuclear proteins labelled at 40 °C, which included a majority of recovered radioactive proteins, were soluble in 1 N NaOH, and can be classified as acidic nuclear proteins. The majority of radioactive leucine was incorporated internally into nuclear proteins, as judged by lack of reactivity with 2,4-dinitrofluorobenzene. Preliminary studies with SDS polyacrylamide gel electrophoresis suggest that some of the radioactive proteins present in the nuclear extract differed from those of the cytosol and mitochondrial fractions. Provided whole cells, mitochondria and endoplasmic reticulum neither contaminated the nuclear pellet nor transferred proteins to that site before or during nuclear isolation, that microorganisms, including PPLO and possibly even viruses capable of causing artefactual incorporation are absent, and that nuclei contain a leucyl tRNA synthetase able to function at 40 °C, the tsHl CHO cell line should provide a valuable experimental system with which to examine the properties of protein synthesis intrinsic to cell nuclei and to elucidate its functions.