Insertion of leader peptidase into the thylakoid membrane during synthesisin a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a tran slation system isolated from chloroplasts to begin to investigate these mec hanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well und erstood for Escherichia coli and for the endoplasmic reticulum. Lep could t hus provide insight into the functional homologies between the different me mbrane systems. Lep was efficiently expressed in the chloroplast translatio n system, and the protein could be inserted into thylakoid membranes with t he same topology as in E. coli cytoplasmic membranes, following the positiv e-inside rule. insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if t hylakoid membranes were present during translation reactions but not when t hylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribos ome nascent chain complexes. These complexes showed a strong, salt-resistan t affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the inserti on of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.