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.

Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.