Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors.
The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.
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Department of Biology, Queen's University, Kingston, Ontario, Canada.
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D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D015723:Gene Library; D008727:Methotrexate / Q000494:pharmacology; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D045322:Photosynthetic Reaction Center Complex Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D003576:Electron Transport Complex IV; D010450:Endopeptidases; D008727:Methotrexate