A Modified Method for Quantification of Cytokinin-Receptor Binding Using Isolated Plant Microsomes Enriched with Cognate Transmembrane Receptors

Cytokinin receptors are key proteins that trigger signaling of cognate classical phytohormones possessing a broad-spectrum biological action. Previously, we developed two test systems to analyze the ligand-binding properties of cytokinin receptors: a heterologous binding assay based on transformed bacteria E. coli, and, later, a more appropriate homologous assay based on microsomes from transiently transformed tobacco (Nicotiana benthamiana Domin.)leaves. In both cases, a genetic construct encoding one or another cytokinin receptor under study, was ectopically expressed. In the present work, we provide a versatile experimental validation for the homologous method and propose a modified (simplified)protocol for obtaining plant microsomes carrying transmembrane proteins to be studied, namely cytokinin receptors. In contrast to the original method, the simplified procedure makes it possible to avoid ultracentrifugation and to save reagents and time when obtaining a microsomal fraction. The microsomes isolated by the modified method were comparable in quantity and quality to a similar fraction obtained in a standard way, in both cases cytokinin specific binding by endogenous receptors was close to zero. Whichever way the microsomes were originally obtained (using an ultra- or preparative centrifuge), they precipitated equally well when subsequently centrifuged at 16?000 g in a benchtop microcentrifuge. All the experimental series allowed us to recommend a wide use of this plant-based binding method, in particular its simplified version, in studies of membrane-bound phytohormone receptors.