Biosynthesis and Assembly of Thylakoid Membrane Proteins in Isolated Chloroplasts from Vicia faba L.: The P700-Chlorophyll a-Protein

Abstract

Summary Isolated Vicia faba chloroplasts were incubated with [ 35 S]methionine in the light, and the newly formed polypeptide products associated with thylakoid membranes were separated by lithium dodecylsulfate polyacrylamide gel electrophoresis (LiDS-PAGE). At least 30 different labeled polypeptides could be distinguished on fluorographs of the gels. Part of them did not comigrate with any stained bands of proteins, but a number of about 15 corresponded in size to preexisting thylakoid components. The inhibition pattern obtained with inhibitors of photophosphorylation (CCCP, DCMU) and translation (cycloheximide, D-threo-chloramphenicol, ribonuclease) indicates that labeling of all these polypeptides occurred in intact chloroplasts only and was energized by photophosphorylation. One of the labeled products comigrated with P700-chlorophyll a-protein (CP I). This labeled protein was precipitated specifically by direct immunoprecipitation and in two-dimensional crossed immunoelectrophoresis using monospecific antibodies against purified CP I apoprotein. Moreover, it was indistinguishable from CP I by the number and size of peptides formed by digestion with S. aureus protease. On this basis, it is suggested that CP I apoprotein was synthesized and assembled by isolated chloroplasts. Polypeptides labeled in isolated chloroplasts comigrated with the α and β subunits of chloroplast coupling factor CF 1 . The patterns of the radioactive fragments obtained from these labeled polypeptides by limited proteolysis corresponded precisely to the patterns of stainable fragments derived from subunits of the purified enzyme. A 32 kd polypeptide was also identified by one-dimensional peptide mapping as a translation product of isolated Vicia faba chloroplasts.