Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.

The insertion of the outer envelope protein Toc34 from chloroplasts was studied. Toc34 was chosen as a model protein because it contains one predicted transmembrane helix at the C-terminus and a large hydrophilic N-terminal located GTPase domain, which is exposed to the cytosol. Unlike proteins located in internal chloroplast compartments, Toc34 neither contains a cleavable presequence nor uses the general import pathway. The protein can insert into the outer envelope of chloroplasts but not into the outer membrane of mitochondria. Using protein-free liposomes we showed that Toc34 is able to insert directly into the lipid bilayer. This insertion is stimulated by GTP and the presence of nonbilayer lipids, but is independent of the presence or absence of charged lipids. The topology of the protein inserted into protein-free liposomes was not exclusively directed by the positive-inside rule but by the size of the hydrophilic domain.

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Post-translational protein import into chloroplasts follows a common route characterised by the need for nucleoside-triphosphates at various steps and two distinct protein import machineries at the outer and inner envelope membrane, respectively. Several subunits of these complexes have been elucidated. In contrast, protein translocation into the chloroplastic outer envelope uses distinct and various but poorly characterised insertion pathways. A topological framework for single-membrane spanning proteins of the chloroplastic outer envelope is presented.