<p>The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of <i>Arabidopsis </i>PreP (<i>At</i>PreP1 and <i>At</i>PreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the <i>At</i>PreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the <i>At</i>PreP1 and <i>At</i>PreP2 are expressed in <i>Arabidopsis</i> in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant <i>At</i>PreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide. </p>

The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of Arabidopsis PreP (AtPreP1 and AtPreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the AtPreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the AtPreP1 and AtPreP2 are expressed in Arabidopsis in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant AtPreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide.

Mitochondria evolved through endosymbiosis of an ancient prokaryote, and subsequently lost most genes to the host genome. In order for mitochondrial proteins to be correctly localized from the host cytosol to the mitochondrial compartments, a complex protein targeting and import machinery has evolved. Key receptor components in the protein translocase complex of the outer mitochondrial membrane, Tom20 and Tom22, recognize proteins to be imported and assist their insertion across the outer membrane. The solution structure of the Tom20 receptor domain from Arabidopsis thaliana was determined by nuclear magnetic resonance spectroscopy, and revealed that this protein has significant structural differences to its functional analogue found in animals and fungi.

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During the transformation of the bacterial endosymbiont into current mitochondria the protein import apparatus had to be created de novo. The reduced mitochondria (mitosomes) of the parasitic protist Giardia intestinalis represent unique cellular model for the examination of these fundamental transport processes. As the main objective of this project I will try to characterize the motor complex, which propels the protein transport, and also the translocation channel in the inner mitosomal membrane. To this aim I will exploit the presence of two membrane components Pam16 and Pam18, which were discovered in our laboratory, and which constitute the functional core of the motor complex. Based on the information from the analogous systems of yeast and mammalian mitochondria, these two components should physically interact with so far unknown translocation channel. In all other eukaryotes this channel is formed by a conserved protein Tim23. The absence of this protein in the genome of G. intestinalis suggests presence of completely novel, or maybe the original-bacterial protein. Having in hand this simplified mitochodrial model the project has potential to bring not only new data in parasite biology but also generate new information on the function and evolution of mitochondrial protein import.

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.

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.

A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an α-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a φχχφφ motif, where φ represents a hydrophobic/aromatic residue and χ represents any amino acid residue. In contrast, neither the amphiphilic helix nor φχχφφ motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction. In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form α-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: In press.</p>