Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport.

Nuclear encoded thylakoid lumen proteins are imported into the chloroplast storma and further directed across thylakoid membranes by lumen targeting domains. Recently, we showed that there are two protein-specific pathways for transport into the lumen. This was unexpected in that lumen targeting domains have similar properties, all containing bacterial signal peptide motifs. Nevertheless, sequence homology analysis suggests that pathway specificity is determined by elements in the lumen targeting domain. To test this, we constructed and analyzed chimeric proteins in which transit peptides from proteins transported by one pathway were fused to the mature domains of proteins directed by the other. We also investigated the transport characteristics of a previously unexamined protein whose pathway was predicted by sequence similarity analysis. Our results confirm that lumen targeting domains contain pathway sorting elements and further indicate that distinct energy and stroma requirements for transport are pathway characteristics, unrelated to the passenger protein. These findings suggest the operation of two mechanistically different translocators.