DOMAINS OF A TRANSIT SEQUENCE REQUIRED FOR IN-VIVO IMPORT IN ARABIDOPSIS CHLOROPLASTS

Nuclear-encoded precursors of chloroplast proteins are synthesized wit h an amino-terminal cleavable transit sequence, which contains the inf ormation for chloroplastic targeting. To determine which regions of th e transit sequence are most important for its function, the chloroplas t uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence we re analyzed. Arabidopsis was used as an experimental system for both i n vitro and in vivo import. The full-length wild-type precursor transl ocated efficiently into isolated Arabidopsis chloroplasts, and upon ex pression in transgenic Arabidopsis plants only mature-sized protein wa s detected, which was localized inside the chloroplast. None of the de letion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal delet ion resulted in a fully defective transit sequence. Two deletions in t he middle region of the transit sequence allowed translocation into th e chloroplast, although with reduced efficiencies. One deletion in thi s region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defecti ve processing. These results allow the dissection of the transit seque nce into separate functional regions and give an in vivo basis for a d omain-like structure of the ferredoxin transit sequence.