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-e volving complex associated with photosystem II are hydrophilic, nuclea r encoded components of the photosynthetic machinery that are all loca ted in the lumen of thylakoid membranes. All four proteins are therefo re imported into chloroplasts and, in addition, translocated across th e thylakoid membrane. They share functionally equivalent, bipartite tr ansit peptides, which are removed in two steps during or after import into the organelle and translocation across the thylakoid membrane, re spectively. The transit peptides lack any homology at the sequence lev el but possess remarkably similar predicted secondary structures. We h ave studied the targeting potential of the authentic precursor molecul es and all possible chimeric combinations generated by a specific, com monly applicable cassette system, which facilitates codon-correct reci procal exchanges of transit peptides and mature parts. An unexpected s pecificity of import and sorting processes was found. All constructs c an be imported into the organelle, though with greatly differing effic iency. 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 t o depend merely on simple interactions of charged or hydrophobic regio ns between protein and membrane but requires an additional quality of information that includes the functional co-evolution of a transit pep tide with its mature protein. Signaling and sorting appear to be essen tial at almost every step of the entire process for proper traffic reg ulation since distinct steps can be impaired (rate-limiting or arreste d) in the individual combinations: the transfer across the envelope me mbranes (e.g. 16/PC) and the interaction with (e.g. 16/33) or the tran slocation across the thylakoid membrane (e.g. 33/23).