Prolines in transit peptides are crucial for efficient preprotein translocation into chloroplasts.

Chloroplasts import many preproteins that can be classified based on their physicochemical properties. The cleavable N-terminal transit peptide (TP) of chloroplast preproteins contains all the information required for import into chloroplasts through Toc/Tic translocons. The question of whether and how the physicochemical properties of preproteins affect TP-mediated import into chloroplasts has not been elucidated. Here, we present evidence that Pro residues in TP mediate efficient translocation through the chloroplast envelope membranes for proteins containing transmembrane domains (TMDs) or proteins prone to aggregation. By contrast, the translocation of soluble proteins through the chloroplast envelope membranes is less dependent on TP prolines. Proless TPs failed to mediate protein translocation into chloroplasts; instead, these mutant TPs led to protein mistargeting to the chloroplast envelope membranes or nonspecific protein aggregation during import into chloroplasts. The mistargeting of TMD-containing proteins caused by Pro-less TPs in wild-type protoplasts was mimicked by wild-type TPs in hsp93-V protoplasts, in which preprotein translocation is compromised. We propose that the physicochemical properties of chloroplast proteins affect protein translocation through the chloroplast envelope, and prolines in TP have a crucial role in the efficient translocation of TMD-containing proteins.