Translational sites of thylakoid membrane proteins. Studies based on selective inhibition of cytoplasmic and chloroplast ribosomes by cycloheximide and chloramphenicol

Abstract

To inhibit chloroplast and cytoplasmic protein synthesis, respectively, excised shoots of 19-day-old Vicia faba plants and 10-day-old Hordeum vulgare plants were incubated with d-threochloramphenicol or cycloheximide in combination with l-[ 35S]methionine. Incubation was performed for 22 h under light subsequent to a 30 min inhibitor pre-incubation without methionine. In order to analyse the translational sites of thylakoid membrane polypeptides, isolated thylakoids were dispersed by sodium dodecyl sulfate and separated by means of two electrophoretic systems. Based on results obtained with electrophoretic system III, 19 of the Vicia faba thylakoid membrane polypeptides sufficiently labeled with l-[ 35S]methionine were inhibited by chloramphenicol and 17 were inhibited by cycloheximide. Nine polypeptides were found to be sufficiently stained but very weakly labeled, indicating low metabolic activity or low methionine content. These polypeptides of low specific radioactivity did not allow analysis of inhibitor effects. Chloramphenicol inhibition included three chlorophyll a-apoproteins. In contrast, the synthesis of three chlorophyll a b- apoproteins , besides some unidentified polypeptides found in different light-harvesting complex preparations of various composition, was severely inhibited by cycloheximide. Comparison of V. faba and H. vulgare thylakoids revealed significant differences in the polypeptide patterns. Both plant species, however, are characterized by three rapidly labeled polypeptides located closely together on the polyacrylamide gels. The identity of these rapidly labeled polypeptide(s) with a herbicide binding protein is discussed.