Synthesis of soluble, thylakoid, and envelope membrane proteins by spinach chloroplasts purified from gradients

Abstract

Intact spinach chloroplasts that had been purified on gradients of silica sol incorporated [ 35S]methionine into soluble and membrane-bound products, using light as the sole energy source. The labeled chloroplasts were lysed osmotically and fractionated on a discontinuous gradient of sucrose into the soluble fraction and the thylakoid and envelope membranes. About 29% of the radioactivity in the chloroplast was recovered in the soluble fraction, 59% in the thylakoid membranes, and 0.1% in the envelope membranes. The products of protein synthesis in the different fractions, as well as in the whole chloroplast, were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. There were two zones of radioactivity in the gels of the soluble fraction, the major zone coincident with the large subunit of ribulose diphosphate carboxylase at a molecular weight of about 50,000. The thylakoid membranes contained five labeled polypeptides, the most active having a molecular weight of about 31,000. The envelope membranes contained a major radioactive component of a molecular weight of about 50,000 and two other minor components.