Post-translational protein targeting requires chaperones and quality control to direct solely insertion-competent membrane proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins into the membrane post-translationally, but mechanisms in the stroma that assist their integration remain largely undefined. The chloroplast chaperonin (Cpn60) bound Plastidic type I signal peptidase 1 (Plsp1), a nuclear-encoded thylakoid transmembrane protein, in the stroma. We investigated how Cpn60 facilitated Plsp1’s thylakoid integration using in vitro targeting assays in isolated chloroplasts and thylakoids. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60 in the stroma. When the Plsp1 sequence that interacted with Cpn60 was removed, Plsp1 did not integrate into the membrane. These results suggested interaction with Cpn60 was an intermediate step in Plsp1’s targeting to the thylakoid. In isolated thylakoids, Plsp1’s integration decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSecA1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases imported Plsp1, while cpSecA1 recognizes free stromal Plsp1 for integration. Plsp1 lost its ability to integrate into thylakoids over time when pretreated with buffer, with all stromal proteins, or with Cpn60. Cpn60 therefore holds unfolded Plsp1 in the stroma until it loses insertion competence, at which point Plsp1 could be routed to an unidentified quality control pathway. We propose that thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.
Coauthors: Kentaro Inoue – University of California, Davis;Steven Theg – University of California, Davis