Stéphane Miras
Identification and characterization of new transport systems of the chloroplast envelope
Published on 22 December 2004
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Thesis presented December 22, 2004
Abstract:
In order to integrate the chloroplast as metabolic compartment into the whole plant cell metabolism, the envelope membranes which surround plastids are the site of many ions and metabolites transport activities. Chloroplasts also import cytoplasmically synthesized precursor proteins from the cytosol. This import process is strictly dependent of an
N-terminal and cleavable transit peptide in the precursor and is achieved by the joint action of the TOC and TIC translocons complexes inserted in the envelope membranes.
A subcellular proteomic approach was developed in the laboratory and lead to the identification of new envelope proteins. Among them, the ceQORH protein was identified. We have demonstrated that the ceQORH protein is the first protein to be targeted to the inner-envelope membrane while lacking such an
N-terminal extension. Moreover, we have demonstrated that the ceQORH protein contains an internal domain coding for the plastid import information, an
N-terminal domain with an unspecific membrane-interacting function and a
C-terminus domain essential for the correct subplasmidial localization of ceQORH in the inner-envelope membrane. Finally,
in vitro import experiments strongly suggest that these central and the
C-terminus domains act concertedly in redirecting the ceQORH protein to a hitherto unknown import site.
The ceQORH protein is related to a range of quinone-oxidoreductases which are largely implicated in electron transfer chains. Therefore, ceQORH could be the first protein component of the redox chain previously described in the chloroplast envelope membranes. We have demonstrated that the chloroplast envelope NADPH:quinone-oxydoreductase activity is correlated to the presence of the ceQORH protein in this membrane. Taken together, these observations are consistent with a potential implication of ceQORH in the redox chain of the inner chloroplast envelope membrane.
Among new chloroplast envelope proteins identified, two putative phosphate transporters (P56/4 and P56/2) with strong sequence homologies to animal phosphate transporters were identified. These proteins could be implicated in plastid uptake of phosphate in order to supply several plastids reactions. We have validated their respective plastid localization using transient expression experiments of these two proteins fused to GFP in
Arabidopsis thaliana cells. Moreover, in order to characterize their functional properties, these proteins were expressed in
S. cerevisiae and in
Xenopus leavis oocytes. Results obtained using these two heterologous expression systems indicate a potential phosphate transport function associated to the P56/4 and P56/2 proteins.
Keywords:
ceQORH, cleavable transit peptide, plastid import, Quinone-oxidoreductase, electron transfer, phosphate transporters, P56/4, P56/2
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