A novel chloroplastic outer membrane-targeting signal that functions at both termini of passenger polypeptides

Several components in the machinery mediating the import of nuclear-encoded chloroplastic precursor proteins have been identified. One of the components, OEP34, is an outer membrane protein and is synthesized at its mature size in the cytosol without a distinguishable chloroplast-targeting signal. To address the question of how components in the transport machinery are imported to chloroplasts themselves, we first identified the chloroplastic outer membrane-targeting signal of OEP34. Using an Arabidopsis homologue of the originally isolated pea OEP34, we show that the outer membrane-targeting signal of OEP34 is located within a 10-amino acid hydrophobic core of the C-terminal membrane anchor. Interestingly, this signal can target a passenger protein to the chloroplastic outer membrane no matter whether it is placed at the N or C terminus of a passenger protein. Proper insertion of fusion proteins into the outer membrane requires in addition the C-terminal hydrophilic region following the hydrophobic core. Furthermore, passenger proteins fused to the C terminus of the targeting/insertion signal were most likely imported into the intermembrane space of the envelope.