Chloroplast signal length requirement reflects the outer membrane and TOC complex dimension
and Purpose: The evolution of an efficient preprotein targeting and translocation system was a central prerequisite for the endosymbiotic integration of a-proteobacteria and cyanobacteria as cellular organelles. Today, it is widely accepted that during evolution most (pre-)proteins destined for these two organelles were equipped with an N-terminal targeting signal for localization. While multiple modes of evolution of these extensions are currently discussed, all evolved signals serve the same
... als serve the same functionforming a signal for targeting to the correct organelle and translocation across both membranes. We aimed to generalize the current idea for the length requirement of the N-terminal extension for efficient translocation. Methods: To explore translocation efficiency in vivo we used protoplasts isolated from different plant sources. We compared the behavior of native and artificial precursor proteins in this plant cell system by fluorescence microscopy. Results: We demonstrate that the minimal length of the N-terminal amino acid stretch in a loosely folded conformation of a precursor of a chloroplast protein is about 60 amino acid residues. This amino acid stretch in a loosely folded state is prerequisite that a preprotein can traverse the outer membrane in vivo. Conclusion: We generalize the evidence that two distinct prerequisites framed the evolutionary process of development of targeting signals for chloroplast translocation. (i) The emerging signal had to be sufficiently distinct to signals existing for targeting to other cellular compartments. (ii) The Nterminal signal had to evolve with physico-chemical properties that serve both purposes: targeting and translocation. With respect to the latter, the length of the unfolded polypeptide is defined by the dimension of the translocon and the resulting distance between the cytosolically exposed receptors acting on the cis side of the membrane and the molecular machinery energizing translocation acting in trans -in the intermembrane space.