Domains of a transit sequence required for in vivo import in Arabidopsis chloroplasts.
Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.
Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. w.a.rensink@bio.uu.nl
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D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002736:Chloroplasts / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D017384:Sequence Deletion
D010940:Plant Proteins