法国国家科学研究中心植物生化研究博士后岗位
来源：丁香人才网
日期：2008-02-18
Institution
Centre National de Recherche Scientifique, CNRS
Position
postdoctoral position in plant biochemistry
Location
Montpellier, France
Date Posted
February 15th 2008
Date Expires
March 25th 2008
Description
Title : Integration of NO3- uptake at the whole plant level
functional roles and post-translational regulations of NRT2.1
The goal of this project is to answer two fundamental questions: (i) Isthe functional complexity of NRT2.1 due to the structural complexityunravelled for this protein? In particular, is the complex of highmolecular weight involved in the control of root development? Is it thenative monomeric form or the truncated protein, which is involved in thefunction of transport? (ii) What are the posttranslational regulationmechanisms of NRT2.1? In particular: is it the C-terminus cleavage thatdrives the activity of the transporter or its role in signalling? Is theprotein regulated by phosphorylation (as suggested by the presence ofpotential phosphorylation sites along the sequence of the protein)?
To answer these questions, the following approaches will be developed incollaboration with the RIO Platform of the Proteomic unit, UR INRA 1199,located on the same campus than our Unit.
(i) Identification of NRT2.1 protein partners and of the end products ofthe C-terminus cleavage
Two strategies will be developed to identify NRT2.1 protein partners:
-Blue Native PAGE will be performed with membranes of transgenic plantsover-expressing NRT2.1 (35S promoter). These plants are available in ourgroup. We thus hope to get larger amount of NRT2.1, which will ease theidentification of its partners using MALDI-TOF-TOF and nano LC-Q-TRAP MS/MS.
-Co-immunoprecipitation of NRT2.1 and its partners using our antibody ortransgenic plants expressing NRT2.1 fused to Myc-tag. These plants arealso available in our group.
To determine the nature of the end products of NRT2.1 C-terminuscleavage; NRT2.1::GFP transgenic plants will be used to purify thecleaved GFP (present in soluble proteins) and identify, usingMALDI-TOF-TOF and nano LC-Q-TRAP MS/MS, NRT2.1 C-terminal part fused tothe N-terminal part of GFP. It will allow to precisely determine thesite of cleavage (a candidate site was found by analysing the sequence).
(ii) Study of NRT2.1 posttranslational regulations by phosphorylation
At first this work will be done using 2D gels with plasma membrane toreveal a possible pH shift for the NRT2.1 protein. This is a classicalapproach used to reveal the existence of phosphorylated forms. Thecomparison between untreated plants and plants subjected to varioustreatments known to affect the activity of the transport system(light/dark, induction by NO3-, repression by NH4+) will also enable tolook at the physiological role of these putative phosphorylations.
Then, immunopurification of NRT2.1 protein using our antibody with35S::NRT2.1 plants and/or transgenic plants transformed with the proteinNRT2.1 fused to an His-tag (available in our group) will enable theidentification of the phosphopeptides and of their sequences usingMALDI-TOF-TOF and nano LC-Q-TRAP MS/M.
(iii) Identification of the physiological roles of NRT2.1 high molecularweight complex and of the posttranslational regulations.
The identification of the proteins interacting with NRT2.1 in the highmolecular weight complex will lead to a reverse genetic approach. Thisshould allow to determine the function of this complex.
The identification of NRT2.1 cleavage site and of putativephosphorylation sites (previous approaches) will be followed by astrategy of directed mutagenesis. We will study in heterologous system(Xenopus oocytes), the transport activity of NRT2.1 mutated proteins.The physiological role in planta of these regulations (NO3- uptake, rootdevelopment) will be study with a pharmacological approach (kinases,phosphatases and proteases inhibitors) and with the production and theanalysis of transgenic plants expressing mutated versions of NRT2.1(C-terminus deletions, mutation of the proteolysis/phosphorylation sites).
The feasibility of this project is supported by (i) the availability ofthe required tools (antibody, transgenic plants), (ii) the collaborationwith UR 1199 for proteomic techniques and (iii) the experience of ourgroup in studying NO3- uptake and root development in A. thaliana.
Depending on the results obtained through the different approaches, themost promising aspects will be favoured.
Contact
http://www.cnrs.fr/
Centre National de Recherche Scientifique, CNRS
Position
postdoctoral position in plant biochemistry
Location
Montpellier, France
Date Posted
February 15th 2008
Date Expires
March 25th 2008
Description
Title : Integration of NO3- uptake at the whole plant level
functional roles and post-translational regulations of NRT2.1
The goal of this project is to answer two fundamental questions: (i) Isthe functional complexity of NRT2.1 due to the structural complexityunravelled for this protein? In particular, is the complex of highmolecular weight involved in the control of root development? Is it thenative monomeric form or the truncated protein, which is involved in thefunction of transport? (ii) What are the posttranslational regulationmechanisms of NRT2.1? In particular: is it the C-terminus cleavage thatdrives the activity of the transporter or its role in signalling? Is theprotein regulated by phosphorylation (as suggested by the presence ofpotential phosphorylation sites along the sequence of the protein)?
To answer these questions, the following approaches will be developed incollaboration with the RIO Platform of the Proteomic unit, UR INRA 1199,located on the same campus than our Unit.
(i) Identification of NRT2.1 protein partners and of the end products ofthe C-terminus cleavage
Two strategies will be developed to identify NRT2.1 protein partners:
-Blue Native PAGE will be performed with membranes of transgenic plantsover-expressing NRT2.1 (35S promoter). These plants are available in ourgroup. We thus hope to get larger amount of NRT2.1, which will ease theidentification of its partners using MALDI-TOF-TOF and nano LC-Q-TRAP MS/MS.
-Co-immunoprecipitation of NRT2.1 and its partners using our antibody ortransgenic plants expressing NRT2.1 fused to Myc-tag. These plants arealso available in our group.
To determine the nature of the end products of NRT2.1 C-terminuscleavage; NRT2.1::GFP transgenic plants will be used to purify thecleaved GFP (present in soluble proteins) and identify, usingMALDI-TOF-TOF and nano LC-Q-TRAP MS/MS, NRT2.1 C-terminal part fused tothe N-terminal part of GFP. It will allow to precisely determine thesite of cleavage (a candidate site was found by analysing the sequence).
(ii) Study of NRT2.1 posttranslational regulations by phosphorylation
At first this work will be done using 2D gels with plasma membrane toreveal a possible pH shift for the NRT2.1 protein. This is a classicalapproach used to reveal the existence of phosphorylated forms. Thecomparison between untreated plants and plants subjected to varioustreatments known to affect the activity of the transport system(light/dark, induction by NO3-, repression by NH4+) will also enable tolook at the physiological role of these putative phosphorylations.
Then, immunopurification of NRT2.1 protein using our antibody with35S::NRT2.1 plants and/or transgenic plants transformed with the proteinNRT2.1 fused to an His-tag (available in our group) will enable theidentification of the phosphopeptides and of their sequences usingMALDI-TOF-TOF and nano LC-Q-TRAP MS/M.
(iii) Identification of the physiological roles of NRT2.1 high molecularweight complex and of the posttranslational regulations.
The identification of the proteins interacting with NRT2.1 in the highmolecular weight complex will lead to a reverse genetic approach. Thisshould allow to determine the function of this complex.
The identification of NRT2.1 cleavage site and of putativephosphorylation sites (previous approaches) will be followed by astrategy of directed mutagenesis. We will study in heterologous system(Xenopus oocytes), the transport activity of NRT2.1 mutated proteins.The physiological role in planta of these regulations (NO3- uptake, rootdevelopment) will be study with a pharmacological approach (kinases,phosphatases and proteases inhibitors) and with the production and theanalysis of transgenic plants expressing mutated versions of NRT2.1(C-terminus deletions, mutation of the proteolysis/phosphorylation sites).
The feasibility of this project is supported by (i) the availability ofthe required tools (antibody, transgenic plants), (ii) the collaborationwith UR 1199 for proteomic techniques and (iii) the experience of ourgroup in studying NO3- uptake and root development in A. thaliana.
Depending on the results obtained through the different approaches, themost promising aspects will be favoured.
Contact
http://www.cnrs.fr/
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