Recombinant Expression of Arabidopsis SNRK1 Kinase in Different Oligomeric Forms

B.E. ROJASE; C.V. PIATTONI; A.A. IGLESIAS

Rosario

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

SAIB

Sucrose non-fermenting-1 related protein kinases 1 (SnRK1s) are heterotrimeric proteins composed of (catalytic) and plus (regulatory) subunits. Members of this kinase family act as central regulators of metabolism. They are generally activated by starvation conditions, generating the trigger of changes in carbon and nitrogen metabolism to maintain energy homeostasis. Kinase action modifies gene expression and enzyme activity through phosphorylation. Arabidopsis genome has two genes codifying for SnRK1 subunit (At3g01090 and At3g29160), three for the subunit (At5g21170, At4g16360 and At2g28060) and two for the subunit (At3g48530 and At1g09020). The latter implies the probable existence of different SnRK1 heteroligomeric forms with distinctive kinetic and/or regulatory properties. Herein we report a protocol to recombinantly express different SnRK1 forms in Escherichia coli after cloning the , , and genes into pDUET vectors. All the combinations (single gene or co-expression of two or three genes) of the SnRK1 proteins were produced and analyzed for activity. We also expressed the kinase (At3g45240) thatphoshorylates (to activate) the SnRK1 catalytic subunit, as well as the mutant mimicking a phosphorylated state. These molecular tools are critical to understand the kinetic, regulatory and target specificity properties of the different SnRK1 assemblies.