Protein import into chloroplasts. The hydrophilic lumenal proteins exhibit unexpected import and sorting specificities in spite of structurally conserved transit peptides.

Plastocyanin and the 16-, 23-, and 33-kDa polypeptides of the oxygen-evolving complex associated with photosystem II are hydrophilic, nuclear encoded components of the photosynthetic machinery that are all located in the lumen of thylakoid membranes. All four proteins are therefore imported into chloroplasts and, in addition, translocated across the thylakoid membrane. They share functionally equivalent, bipartite transit peptides, which are removed in two steps during or after import into the organelle and translocation across the thylakoid membrane, respectively. The transit peptides lack any homology at the sequence level but possess remarkably similar predicted secondary structures. We have studied the targeting potential of the authentic precursor molecules and all possible chimeric combinations generated by a specific, commonly applicable cassette system, which facilitates codon-correct reciprocal exchanges of transit peptides and mature parts. An unexpected specificity of import and sorting processes was found. All constructs can be imported into the organelle, though with greatly differing efficiency. On the other hand, the lumen-targeting parts are essential but not in all cases sufficient for correct intraorganellar routing. This implies that translocation across the thylakoid membrane appears not to depend merely on simple interactions of charged or hydrophobic regions between protein and membrane but requires an additional quality of information that includes the functional co-evolution of a transit peptide with its mature protein. Signaling and sorting appear to be essential at almost every step of the entire process for proper traffic regulation since distinct steps can be impaired (rate-limiting or arrested) in the individual combinations: the transfer across the envelope membranes (e.g. 16/PC) and the interaction with (e.g. 16/33) or the translocation across the thylakoid membrane (e.g. 33/23).