AMINO-TERMINAL AND HYDROPHOBIC REGIONS OF THE CHLAMYDOMONAS-REINHARDTII PLASTOCYANIN TRANSIT PEPTIDE ARE REQUIRED FOR EFFICIENT PROTEIN ACCUMULATION IN-VIVO

Nucleus-encoded chloroplast proteins of vascular plants are synthesize d as precursors and targeted to the chloroplast by stroma-targeting do mains in N-terminal transit peptides. Transit peptides in Chlamydomona s reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochond rial and chloroplast targeting sequences. To examine Chlamydomonas tra nsit peptide function in vivo, deletions were introduced into the tran sit peptide coding region of the petE gene, which encodes the thylakoi d lumen protein plastocyanin (PC). The mutant petE genes were introduc ed into a plastocyanin-deficient Chlamydomonas strain, and transforman ts that accumulated petE mRNA were analyzed for PC accumulation. The m ost profound defects were observed with deletions at the N-terminus an d those that extended into the hydrophobic region in the C-terminal ha lf of the transit peptide. PC precursors were detected among pulse-lab eled proteins in transformants with N-terminal deletions, suggesting t hat these precursors cannot be imported and are degraded in the cytoso l. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defec tive in lumen translocation and/or processing. Thus, despite its short er length, the bipartite nature of the Chlamydomonas PC transit peptid e appears similar to that of lumen-targeted proteins in vascular plant s. Analysis of the synthesis, stability, and accumulation of PC specie s in transformants bearing deletions in the stroma-targeting domain su ggests that specific regions probably have distinct roles in vivo.