Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins. Plant 14-3-3 proteins interact specifically with wheat germ lysate-synthesized chloroplast precursor proteins and require an intact phosphorylation motif within the transit sequence. Chloroplast precursor proteins do not interact with 14-3-3 when synthesized in the heterologous reticulocyte lysate. In contrast, a precursor protein destined for plant mitochondria was found to be associated with 14-3-3 proteins present in the reticulocyte lysate but not with 14-3-3 from wheat germ lysate. This indicates an unrecognized selectivity of 14-3-3 proteins for precursors from mitochondria and plastids in plants in comparison to fungi and animals. The heterooligomeric complex has an apparent size of 200 kD. In addition to the precursor protein, it contains 14-3-3 (probably as a dimer) and a heat shock protein Hsp70 isoform. Dissociation of the precursor complex requires ATP. Protein import experiments of precursor from the oligomeric complex into intact pea chloroplasts reveal three- to fourfold higher translocation rates compared with the free precursor, which is not complexed. We conclude that the 14-3-3-Hsp70-precursor protein complex is a bona fide intermediate in the in vivo protein import pathway in plants.

Chloroplasts are believed to have originated from a photosynthetic, prokaryotic ancestor. As the result of endosymbiotic evolution, most of the genes of the endocytobiont were displaced to the host nucleus. Today's chloroplasts must import most of their proteins from the cytosol as precursors. Oligomeric protein complexes in the chloroplast outer and inner envelope membranes are responsible for the specific recognition and membrane translocation of precursor proteins. The translocon at the outer membrane of chloroplasts and the inner membrane of chloroplasts act jointly during the import process. Several translocon subunits have been partially characterized in their molecular structure and function. Initial evidence indicates the prokaryotic origin of some chloroplast translocon components.

The protein import complex of the chloroplastic outer envelope (Toc-complex) contains a prominent subunit of 86 kDa molecular weight (Toc86). Toc86 was identified as a putative precursor receptor. The Arabidopsis genome sequencing project indicates that Toc86 represents only a proteolytic fragment of a larger polypeptide of 160 kDa. The 160-kDa protein, which we name Toc160, is only present in significant amounts in pea chloroplasts isolated under stringent conditions. The capacity of chloroplasts to import an in vitro translated precursor protein correlates well with the integrity of Toc160. We conclude that Toc160 is still a bonafide subunit of the protein import machinery of chloroplasts.

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.