Recombinant assembling of Arabidopsis thaliana SnRK1 kinase complexes

ROJAS, BRUNO E; PIATTONI, CLAUDIA V.; IGLESIAS, ALBERTO A.

Mar del Plata

Congreso; XXX REUNION ANUAL DE LA SOCIEDAD ARGENTINA DE FISIOLOGÍA VEGETAL; 2014

Sociedad Argentina de Fisiología Vegetal

The sucrose non-fermenting-1 related protein kinase 1 (SnRK1s) (evolutionarily related with yeast´s SNF1 and mammalian AMPK) are heterotrimeric metabolic sensors composed of α (catalytic) and β plus γ (regulatory) subunits. These kinases share the ancestral function as central regulators of metabolism, being activated by starvation conditions and triggering changes in carbon and nitrogen metabolism to maintain energy homeostasis by modifying gene expression and enzyme activity through phosphorylation. Arabidopsis genome has two genes codifying for the α subunit (At3g01090 and At3g29160), three for the β subunit (At5g21170, At4g16360 and At2g28060) and two for the γ subunit (At3g48530 and At1g09020) of SnRK1. The latter implies the possibility of the existence of different SnRK1 heteroligomeric forms. The kinetic and/or regulatory properties of such distinct kinases have not been reported. We designed a protocol to recombinantly generate different SnRK1 forms. All the α, β, and γ genes were cloned into pDUET vectors, to express them in Escherichia coli BL21DE3 cells. All the combinations (separately or co-expression between two or three) of the SnRK1 proteins were produced and analyzed for activity. We also expressed the kinase that phoshorylates (to activate) the SnRK1 catalytic subunit (At3g45240), as well as an α mutant version mimicking its phosphorylation.