The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid.

KEY MESSAGE: The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP-GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.