Development of\na Versatile Plant-Derived Mitochondrial\nTargeting Sequence Based on a Reporter Protein Sorting Analysis and\nBiological Information

Methods for the delivery of exogenous substances to specific\norganelles\nare important because each organelle functions according to its own\nrole. Specifically, mitochondria play an important role in energy\nproduction. Recently, plant mitochondrial transformation via delivery\nmethods to mitochondria has been actively researched. Mitochondrial\ntargeting sequences (MTSs) are essential for transporting bioactive\nmolecules, such as nucleic acids, to mitochondria. However, the selectivity\nand efficacy of MTSs as carrier molecules in plants are not yet sufficient.\nIn this study, we developed an effective MTS in plants via a quantitative\ncomparison of the targeting functions of several MTSs. The presequence\nof HSP60 from Nicotiana tabacum, which\nis highly similar to that of several other model plants, showed high\nmitochondrial-targeting ability among the MTSs tested. This result\nsuggests the applicability of the HSP60 presequence for MTSs in various\nplants. We further investigated this HSP60 presequence through stepwise\nshortening on the basis of secondary structure prediction, aiming\nto simplify synthesis and increase the solubility of the peptides.\nAs shown by assessment of the mitochondrial targeting ability, the\n15 residues from the N-terminus of the HSP60 presequence for the MTS,\nwhich is particularly conserved among various model plants, retained\na targeting efficacy comparable to that of the full-length HSP60 presequence.\nThis developed sequence from the HSP60 sequence is a promising MTS\nfor transfection into plant mitochondria.