Séminaire IBIP
Les séminaires ont lieu sur le Campus Montpellier SupAgro/INRA de La Gaillarde (2, place P. Viala Montpellier)

Jeudi 8 avril 2010
Amphi 208 (Cœur d’Ecole) à 14h

How to get to know a transport protein: Structure, function and regulation of the NO3-/H+ exchanger AtCLCa

Steffi Wege
ISV, CNRS Gif-sur-Yvette

The Arabidopsis thaliana CLCa belongs to the ChLoride Channel (CLC) family of anion transport proteins. Unlike the mammalian members of the family, AtCLCa is most selective for nitrate and not for chloride. It mediates the accumulation of nitrate into the vacuole by a NO3-/H+ exchanger mechanism and clca knock out mutants under accumulate nitrate on the whole plant level. This difference in selectivity is accompanied by a single amino acid change in the otherwise conserved selectivity filter. We could show that an exchange of one amino acid turns AtCLCa into a chloride transporter and abolishes its function of nitrate accumulation in planta. GUS expression studies showed that AtCLCa is expressed throughout the plant, with a strong expression in stomata guard cells. Phenotype analysis of clca ko mutants demonstrate a role of AtCLCa not only in nitrate accumulation but also in stomata movement. We were then interested how the activity of AtCLCa is regulated in the different cell types in which it is expressed. Other transport proteins have been shown to be regulated by phosphorylation and also AtCLCa was found to be phosphorylated in vivo. We identified two possible regulatory protein partners that belong to a specific class of kinases, the SnRKs. One of the two kinases, OST1 (SnRK2.6), is strongly expressed in guard cells and is well integrated in ABA signalling. The other kinase, AKIN10 (SnRK1.1), is expressed throughout the plant and can inhibit a component of nitrate metabolism, the nitrate reductase. We verified that both candidate kinases are able to phosphorylate AtCLCa in vitro and interact with it in vivo. Using the Patch-Clamp technique on isolated vacuoles we could find a direct influence of the phosphorylation on the activity of AtCLCa.

Contact : Michael Wudick

Contacts IBIP :
Sabine Zimmermann
Marc Lepetit
Christine Granier
Corinne Dasen
Chantal Baracco