Discovering the functions of the acidic domain of Toc159

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.

Thèse de doctorat : Université de Neuchâtel, 2014