In vitro reconstitution of insertion and processing of cytochrome f in a homologous chloroplast translation system.

Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated post-translationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.