<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.

Most chloroplast proteins are encoded in the nucleus and translated in the cytosol with an N-terminal transit peptide, which facilitates recognition by the receptors of the translocon at the outer membrane of chloroplasts (Toc). The Toc159 family of receptors in Arabidopsis thaliana are the primary chloroplast preprotein receptors. Members of this family differentially associate with either atToc33 or atToc34 (“at” designates the species of origin, Arabidopsis thaliana) to form structurally and functionally distinct Toc complexes; atToc159/33-containing complexes import photosynthetic preproteins, and atToc132(120)/34-containing complexes import non-photosynthetic, plastid house-keeping proteins. The Toc159 receptors are most variable in their N-terminal A-domain, suggesting that this domain may contribute to their functional specificity. The A-domain has structural properties characteristic of intrinsically unstructured protein (IUP) domains, including an abundance of acidic amino acid residues, aberrant mobility during SDS-PAGE and sensitivity to proteolysis. The overall objective of this study was to gain insight into the function of the A-domain. First, to investigate the role of the A-domain in the assembly of structurally distinct Toc complexes, full-length, truncated and domain-swapped variants of atToc159 and atToc132 were targeted in vitro to chloroplasts isolated from wild type (WT) Arabidopsis, and atToc33 and atToc34 null mutants (ppi1 and ppi3, respectively). Insertion of atToc132 was less efficient than atToc159, and was not affected by the removal or swapping of the A-domain. In contrast, removal of the A-domain of atToc159 resulted in decreased insertion, most notably into ppi1 chloroplasts, suggesting that the A-domain is important for insertion, especially into atToc34-containing complexes. These results indicate that the A-domain does play a role in targeting, and may also suggest different roles for the A-domain in targeting of atToc159 and atToc132. Second, a structural analysis of the A-domain of atToc132 and atToc159 was performed using CD and fluorescence spectroscopy to gain insight into their potential function(s). The A-domains were found to be unstructured at physiological pH, and their secondary structure increased with increasing temperature and decreasing pH, which are characteristics of IUPs. IUPs are commonly involved in protein-protein interactions, and their unstructured nature may suggest a role for the A-domains in binding transit peptides, accounting for the ability of the Toc159 receptors to differentially distinguish between a large number of diverse transit peptides that possess low sequence conservation.

Most chloroplast proteins are encoded in the nucleus and translated in the cytosol with an N-terminal transit peptide, which facilitates recognition by the receptors of the translocon at the outer membrane of chloroplasts (Toc). The Toc159 family of receptors in Arabidopsis thaliana are the primary chloroplast preprotein receptors. Members of this family differentially associate with either atToc33 or atToc34 (“at” designates the species of origin, Arabidopsis thaliana) to form structurally and functionally distinct Toc complexes; atToc159/33-containing complexes import photosynthetic preproteins, and atToc132(120)/34-containing complexes import non-photosynthetic, plastid house-keeping proteins. The Toc159 receptors are most variable in their N-terminal A-domain, suggesting that this domain may contribute to their functional specificity. The A-domain has structural properties characteristic of intrinsically unstructured protein (IUP) domains, including an abundance of acidic amino acid residues, aberrant mobility during SDS-PAGE and sensitivity to proteolysis. The overall objective of this study was to gain insight into the function of the A-domain. First, to investigate the role of the A-domain in the assembly of structurally distinct Toc complexes, full-length, truncated and domain-swapped variants of atToc159 and atToc132 were targeted in vitro to chloroplasts isolated from wild type (WT) Arabidopsis, and atToc33 and atToc34 null mutants (ppi1 and ppi3, respectively). Insertion of atToc132 was less efficient than atToc159, and was not affected by the removal or swapping of the A-domain. In contrast, removal of the A-domain of atToc159 resulted in decreased insertion, most notably into ppi1 chloroplasts, suggesting that the A-domain is important for insertion, especially into atToc34-containing complexes. These results indicate that the A-domain does play a role in targeting, and may also suggest different roles for the A-domain in targeting of atToc159 and atToc132. Second, a structural analysis of the A-domain of atToc132 and atToc159 was performed using CD and fluorescence spectroscopy to gain insight into their potential function(s). The A-domains were found to be unstructured at physiological pH, and their secondary structure increased with increasing temperature and decreasing pH, which are characteristics of IUPs. IUPs are commonly involved in protein-protein interactions, and their unstructured nature may suggest a role for the A-domains in binding transit peptides, accounting for the ability of the Toc159 receptors to differentially distinguish between a large number of diverse transit peptides that possess low sequence conservation.