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Discovering the functions of the acidic domain of Toc159
Auteur(s)
Munusamy Lakshmanan, Ashok
Editeur(s)
Date de parution
2014
Résumé
Preprotein import from the cytosol is a key mechanism in chloroplast biogenesis. Hundreds of different preproteins take an import route involving the chloroplast surface receptor Toc159. Toc159 has three domains A (acidic), G (GTPase) and M (membrane insertion). The N-‐terminal, cytosolic A-‐domain has been shown to contribute to preprotein specificity of Toc159 (Toc159A). This thesis began with specific questions to address the existence of hyperphosphorylated and soluble Toc159A as a separate protein in the cytosol and aims at expanding our understanding of Toc159-‐dependent chloroplast protein import.<br>In its first part, this thesis demonstrates the dual localization of Toc159A both in the cytosol and at the chloroplast envelope using TAP (tandem affinity purification)-‐tagged Toc159A and Toc159A-‐YFP (yellow fluorescent protein). Blue native polyacrylamide gel electrophoresis BN-‐PAGE in combination with Western blotting identified Toc159A-‐TAP complexes. However, the weak stability of the complexes required stabilization of the complexes by chemical crosslinking by formaldehyde prior to purification. Complexes identified by BN-‐PAGE were confirmed by formaldeyhde crosslinking. Complexes were affinity purified and analysed by mass spectrometry. We identified putative interacting proteins that could be classified into three main categories. 1) Chloroplast targeted Toc159-‐dependent preproteins 2) two kinases predicted to be located at the chloroplast envelope and in the cytoplasm, 3) cytosolically localized Hsp70 and Calmodulin. Upon confirmation, all of these offer exciting future research perspectives.<br>The second part of the thesis focuses on in vitro experiments to understand possible roles of Toc159A in chloroplast protein import and Toc159A phosphorylation. Two hypotheses were tested:<br>1) First hypothesis: direct binding of the pSSU (pre-‐small subunit of Rubisco) preprotein to Toc159A prior to import into the organelle. This hypothesis was tested by adding recombinant A-‐domain to in vitro import assays. Inhibition of import at the earliest stages suggested an effect on the kinetics of import. <br>2) Second hypothesis: regulation of ribosomal translational activity by the A-‐domain. This hypothesis was tested by adding phosphorylatable or non-‐phosphorylatable A-‐domain to in vitro translation reactions. Phosphorylatable Toc159A had a positive effect on the in vitro translation of an early developmental stage specific preprotein pSSU but negatively affected the late developmental stage specific preprotein pTic40. This suggests that Toc159 may directly affect the synthesis of preproteins that take an import route required early in development. While these results are in need of further experimentation, they suggest a novel mechanism for coupling preprotein translation to chloroplast protein import.
Notes
Thèse de doctorat : Université de Neuchâtel, 2014
Identifiants
Type de publication
doctoral thesis
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