Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation.
Nucleus-encoded chloroplast proteins that reside in the thylakoid lumen are synthesized as precursors with bipartite transit peptides that contain information for uptake and intra-chloroplast localization. We have begun to apply the superb molecular and genetic attributes of Chlamydomonas to study chloroplast protein import by creating a series of deletions in the transit peptide of plastocyanin and determining their effects on translocation into isolated Chlamydomonas chloroplasts. Most N-terminal mutations dramatically inhibited in vitro import, whereas replacement with a transit peptide from the gamma-subunit of chloroplast ATPase restored uptake. Thus, the N-terminal region has stroma-targeting function. Deletions within the C-terminal portion of the transit peptide resulted in the appearance of import intermediates, suggesting that this region is required for lumen translocation and processing. Thus, despite its short length and predicted structural differences, the Chlamydomonas plastocyanin transit peptide has functional domains similar to those of vascular plants. Similar mutations have been analyzed in vivo by transforming altered genes into a mutant defective at the plastocyanin locus (K. L. Kindle, manuscript in preparation). Most mutations affected in vitro import more severely than plastocyanin accumulation in vivo. One exception was a deletion that removed residues 2-8, which nearly eliminated in vivo accumulation but had a modest effect in vitro. We suggest that this mutant precursor may not compete successfully with other proteins for the translocation pathway in vivo. Apparently, in vivo and in vitro analyses reveal different aspects of chloroplast protein biogenesis.
Lawrence
S D
SD
Plant Science Center, Cornell University, Ithaca, New York 14853, USA.
Kindle
K L
KL
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002696:Chlamydomonas / Q000378:metabolism*; D008969:Molecular Sequence Data; D010970:Plastocyanin / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism
D002352:Carrier Proteins; D011498:Protein Precursors; D000255:Adenosine Triphosphate; D010970:Plastocyanin