Involvement of Transit Peptide Aromatic Residues in Precursor Interaction(s) with the Chloroplast Import Apparatus

The import of nuclear-encoded preproteins into chloroplast is mediated by the transit peptide located in the N-terminus of the preproteins. A semi-conserved FGLK motif is identified in most of the transit peptides bioinformatically, despite the lack of homology in the primary sequence. To investigate the role of phenylalanines in the FGLK motifs of SStp in the interaction with outer envelope import apparatus, mutations were made on the two motifs found in prSSU from tobacco: F26TGLK and F35PVSRK. These mutations include F→W, F→S, and F→A in both of the motifs. The WT precursor (prSSU), 6 single prSSU mutants, 9 double prSSU mutants and mature protein were expressed and purified from E. coli. The activities of 13 out of 15 mutant proteins are then tested as compeptivie inhibitors of the import of 35S-prSSU. The increase in IC50 indicated that both phenylalanines at position 26th and 35th are important for the import process. Membrane disruption assay showed that all these mutants have slightly reduced membrane activity compared to wild type, suggesting that interaction with membrane lipid components is secondary to other interactions with proteinaceous component in the early event of the import process. Tryptophan fluorescence assay using the transit peptide mutants F26W-SStp and F35W-SStp demonstrated that the two loosely conserved FGLK motifs have different positions when exposed to a hydrophobic environment, indicative of different roles they might have in the recognition/binding process.