SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic β and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. TheArabidopsis thalianagenome contains homologues of each subunit type, and, in addition, two atypical subunits, β3 and βγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. TheAtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique βγ subunit and one of three non-catalytic β subunits: β1, β2 or the plant-specific β3 isoform. Thus, theAtSnRK1 heterotrimers contain the atypical βγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T–loop (pThr175/176) within both catalytic a subunits. However,AtSnRK1 is insensitive to AMP and ADP, and is resistant to T–loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the β1, β2 and βγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK bindsin vitro.These data clearly demonstrate thatAtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.Significance Statement
Here we show that SnRK1, an energy-sensing enzyme found in plants, can exist as six different isoenzymes. Compared to AMP-activated protein kinase, the mammalian homologue, SnRK1 is not affected by phosphorylation, nucleotides or carbohydrates. This knowledge will contribute to understanding the role of SnRK1 in how plants cope with stress and maintain crop yield.