Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein.
The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH2-terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.
Silva-Filho
M D
MD
Unité de Biochimie Physiologique, Université catholique de Louvain, Place Croix du Sud, 2-20, B-1348 Louvain-la-Neuve, Belgium.
Wieërs
M C
MC
Flügge
U I
UI
Chaumont
F
F
Boutry
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D030821:Plants, Genetically Modified
D060365:Chloroplast Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D015500:Chloramphenicol O-Acetyltransferase