AbstractPost-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1’s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 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 cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.One-sentence summaryThe chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.