Folding states of transit peptides and mature peptides in the cytosol: implications for the import-competent state of preproteins.
Most plastid proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins (or precursors). These preproteins consist of N-terminal TPs (transit peptides) followed by MPs (mature peptides, which ultimately fold into their mature conformations inside plastids). The preproteins are transported across plastid membranes via the TOC/TIC complex. Since the channel of TOC/TIC is too narrow to accommodate fully or highly folded preproteins, it has been widely assumed that preproteins are maintained in an unfolded or partly folded state in the cytosol to facilitate their import. However, whether a dedicated mechanism exists to actively inhibit MP folding in the cytosol remains unclear. Here we show that such a mechanism may not exist, because MPs can acquire folded conformations while still in the cytosol. Surprisingly, we found that two preproteins with identical amino-acid sequences - one produced by de novo synthesis and the other by protease-mediated cleavage - exhibited different organelle-targeting abilities, suggesting that the primary structures of TPs are not sufficient to direct exclusive plastid import and that TPs need to fold into a specific conformation to function properly. Based on these results, we propose that the faster folding of TPs, relative to downstream MPs, plays an important role in maintaining preproteins in an import-competent state.