The biogenesis of the cyanellae of Cyanophora paradoxa . - II. Pulse-labelling of cyanellar polypeptides in the presence of transcriptional and translational inhibitors

Abstract

Cycloheximide and chloroamphenicol, specific inhibitors of protein translation in the cytoplasmic and cyanellar compartments, respectively, of Cyanophora paradoxa , have been employed in 30 min pulse-labelling experiments by using [NaH- 14 C]O 3 to label total cell proteins in vivo . Cyanellae purified from host cell lysates were separated into soluble and thylakoid fractions and analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to determine the distribution of radio-activity in the cyanellar polypeptides. Analysis of the autoradiograms of electrophoretically resolved proteins of the cyanellae indicates that about 70% of the total number of cyanellar proteins visualized in the controls are synthesized on cytoplasmic ribosomes. The majority (81%) of the soluble cyanellar proteins appear to be cytoplasmically synthesized. In contrast, the majority (70%) of the thylakoid proteins are synthesized within the cyanellae. The observations also suggest that the polypeptides synthesized within the cyanellae include species that are the most abundant and rapidly turned over. A number of the polypeptides previously identified have now been characterized with regard to their sites of synthesis. In addition, we report on labelling experiments involving rifampicin, a specific inhibitor of cyanellar transcription, which indicate that different mRNAs within the cyanellae have markedly different stabilities.