Detection of a Precursor Polypeptide of the Rapidly-Synthesized 32,000-Dalton Thylakoid Protein in Spinach Chloroplasts

Spinach chloroplast RNA was translated in a wheat germ cell-freesystem in the presence of ³⁵S]methionine or ³H]lysine, andthe products were analyzed by SDS polyacrylamide gel electrophoresisand fluorography. A polypeptide with molecular mass of 2,000-Dalarger than the 32,000-Da thylakoid protein was detected asa major product labeled by ³⁵S]methionine but not by ³H]lysine.Peptide mapping of this polypeptide showed a pattern very closeto that of the 32,000-Da protein synthesized in isolated chloroplasts.A better separation of this polypeptide from the 32,000-Da proteinwas observed in the electrophoresis on polyacrylamide gel includingurea at 8 M. Pulse-labeling of the isolated chloroplasts showedthe occurrence of the larger molecular weight form, which wasconverted to the mature size by a chasing incubation with coldmethionine. These results suggested that the 32,000-Da proteinof spinach is translated primarily as a high molecular weightprecursor in the chloroplasts, as has been reported for otherplant species. (Received March 30, 1985; Accepted April 23, 1985)