Ribosomes pause at specific sites during synthesis of membrane-bound chloroplast reaction center protein D1

Photosynthetic reaction center protein D1 contains five membrane-spanning α-helices which form binding sites for pheophytin, chlorophyll, carotenoids, quinone, Fe²⁺, and probably Mn²⁺. D1 translation intermediates of 15 to 28 kD were detected when isolated chloroplasts were pulse-labeled with [³⁵S]methionine. The D1 translation intermediates were associated with membrane polysemes and can be chased into full length D1. The sites of translation pausing were determined by mapping the distribution of ribosomes on D1 mRNA using toeprint analysis. Clusters of toeprint signals were generated by D1 mRNA associated with membranes but not by D1 mRNA in nonpolysomal fractions of the soluble phase or phenol-extracted mRNA. The distribution of ribosomes on D1 mRNA determined by toeprint analysis was consistent with D1 translation intermediates observed with pulse-labeling. Ribosome pausing may facilitate co-translational binding of cofactors such as chlorophyll to D1 and aid the integration of D1 into thylakoid membranes.