A role for the carbohydrate-binding module (CBM) in regulatory SnRK1 subunits: the effect of maltose on SnRK1 activity

SnRK1 is a protein kinase complex that is involved in several aspects of plant growth and development. There are published data indicative of a participation of SnRK1 in the regulation of the synthesis and degradation of starch, although the molecular mechanism is not known. In this work, we performed electron microscopy to explore the in vivo localization of the regulatory and catalytic subunits that constitute the SnRK1 complex. The results indicated that all the subunits are present in the chloroplast and, in particular, the SnRK1 beta gamma and SnRK1 beta 3 subunits are associated with starch. Furthermore, the regulatory subunits bind maltose, a relevant product of starch degradation. The kinase activity of immunoprecipitated complexes containing the beta gamma regulatory subunit was positively regulated by maltose only in the complexes obtained from Arabidopsis leaves collected at dusk. Recombinant complexes with the SnRK1 alpha 1 catalytic subunit, SnRK1 beta gamma and three different beta subunits showed that maltose only had an effect on a complex formed with the beta 3 subunit. Truncation of the CBM domain form SnRK1 beta gamma abolished the maltose activation of the complex and the activity was significantly reduced, indicating that the CBM is a positive regulator of SnRK1. A model of the SnRK1 alpha 1/beta gamma/beta 3 complex suggests the presence of two putative maltose-binding sites, both involving ligand interactions with the beta gamma subunit and the alpha subunit.