<p>  </p> <p>Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all plants and certain algae. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their function. The translocon at the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components in plants: Toc75, Toc33/34 and Toc159. Our overall goal is to elucidate the architecture of the TOC complex to gain mechanistic insights into protein translocation into chloroplasts. However, the major bottleneck preventing structure determination of the TOC complex has been the inability to produce or isolate the complex to sufficient yields and purity for structural studies. We began by using phage display to screen for synthetic antigen-binding fragments (sABs) that bind to the soluble POTRA domains of Toc75 from both <em>Arabidopsis thaliana</em> and <em>Pisum sativum</em>. We then characterized the POTRA-sAB interactions using size-exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS), isothermal titration calorimetry (ITC), and X-ray crystallography. Finally, we show that we can use an affinity tagged sAB to isolate the TOC complex directly from pea biomass. This study has paved the way for high-resolution structural studies of the TOC complex from plants to understand protein translocation mechanisms. </p>

The majority of chloroplast proteins are nuclear-encoded and post-translationally imported into the chloroplast. These newly imported proteins are translocated from the cytosolic compartment to the stroma by the Translocons of the Outer/Inner membranes of Chloroplast (TOC/TIC). In order to understand protein transport across the chloroplast outer membrane, it is crucial to investigate the structure and function of these complexes. The TOC complex is composed of the beta-barrel channel protein Toc75 and the GTPase receptors Toc34 and Toc159. Toc75 is a member of the OMP85 (Outer Member Protein, 85 kDa) superfamily. Other proteins of the OMP85 superfamily also exist in Gram-negative bacteria and mitochondria. The members of this family contain a C-terminal transmembrane beta-barrel and a soluble N-terminus with a varying numbers of POTRA (POlypeptide TRansport Associated) domains. The recent crystal structures of the POTRA domains of Gram-negative bacteria reveal that these domains are localized in the periplasmic side. This thesis identifies the orientation of the POTRA domains as being localized in the cytosol and provides initial evidence for their involvement in the protein import. Three POTRA domains of psToc75 were identified, purified in E. coli and characterized by MALDI-TOF mass spectrometry and circular dichroism. Using variety of immunofluorescence methods, such as flow cytometry and LSCM, the topology of the POTRA domains was investigated. Chloroplast agglutination assays were used to assess the location of immuno-reactive fragments of the POTRA domains, which supported the results from the flow cytometry and LSCM. Finally, thermolysin was used to probe the surface of the isolated intact chloroplasts. Proteolytic digestion along with the data obtained from flow cytometry, LSCM and agglutination assays suggested the orientation of the N-terminal POTRA domains facing the cytosol, followed by a C-terminal beta-barrel domain. The import competence of individual POTRA domains was determined by in vitro chloroplast import and binding competition assays. POTRA1 inhibited the binding of the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts, while POTRA3 inhibited the import of radiolabeled precursors into isolated chloroplasts; however, in both assays, the inhibition of precursor binding and import was to a lesser extent than non-labeled prSSU.

During chloroplast formation via endosymbiosis most of the plastid genome was transferred to the host nuclear genome. Genomic and proteomic analysis suggests that >95% of the original plastid proteome is now encoded in the nucleus, and these now cytosolically fabricated proteins require a post-translational transport pathway back into the organelle. This process is not well understood, yet it has been shown to involve translocons at the outer and inner envelope of the chloroplast membranes (TOC & TIC). These translocons interact with a cleavable N-terminal extension of between 20 and 100 residues on chloroplast-bound precursor proteins known as the transit-peptide. Precursor proteins pass through the outer membrane via the outer chloroplast membrane beta-barrel, Toc75. In addition to containing a transmembrane β-barrel, Toc75 also contains three polypeptide transport (POTRA) domain repeats at the N-terminus. Despite widespread occurrence the role of POTRAs is poorly understood. One possibility is that they function to promote either homo- or heterotypic protein:protein interactions. To investigate these possibilities, we modeled the psToc75 POTRA domains and purified recombinant POTRA domains. POTRA1, POTRA3, and POTRA1-3 have been used to investigate interactions. Homotypic POTRA interactions have been supported by crosslinking experiments and analytical ultra centrifugation (AUC). Crosslinking data shows POTRA1 and POTRA3 undergo oligimerization. AUC suggests that POTRA1 may homodimerize. Heterotypic interactions have been studied via pull-down assays, crosslinking, and AUC and demonstrate that POTRA1 and POTRA3 interact with transit peptide. Soluble POTRA1-3 seems to stimulate precursor protein import into isolated chloroplasts in an import assay. The role of POTRAs in guiding TOC assembly by homodimerization is being investigated, and experiments to establish how POTRAs aggregate are underway.