We sought to determine the structural features involved in the processing of the mitochondrial F1-ATPase β-subunit (F1β) presequence (54 residues) from Nicotiana plumbaginifolia. The cleavage efficiency of F1β presequence mutants linked to the green fluorescent protein (GFP) was evaluated in vivo in tobacco by in situ microscopy and Western blotting. The residue at position −1 (Tyr) was required to be an aromatic residue and the residue at position +2 (Thr) was found to be important for F1β processing, while, unexpectedly, changing the distal (Arg-15) and proximal (Arg-5) arginine residues did not strongly reduce processing. In addition, results also supported the requirement of a helical structure around the cleavage position. Sequencing of the mature form of a precursor containing the first 30 residues of the F1β presequence linked to GFP revealed the presence of a cryptic cleavage site between residues 26 and 27, which showed the features of a classical mitochondrial processing site, suggesting dual processing of the F1β presequence. In vitro processing confirmed these data and showed that processing was sensitive to o-phenanthroline, thus catalyzed by mitochondrial processing peptidase.
