Evidences for Preferential Localization of Cytokinin Receptors of Potato in the Endoplasmic Reticulum

Abstract

Cytokinins, classical plant hormones, are recognized by the cell through receptors—hybrid sensor histidine kinases, multidomain transmembrane proteins. The character of the hormonal effect on the cell and the mode of the hormonal signal transduction to the primary targets depends on the subcellular localization of the receptors. Recently, we cloned and investigated a family of cytokinin receptors from a new model plant, potato Solanum tuberosum L. Two typical representatives of this family, StHK2a and StHK4b, were selected to study their subcellular localization. In the current study, total membranes isolated from tobacco leaves, in which the selected recombinant potato receptors fused to GFP-reporter were transiently expressed, were fractionated by aqueous two-phase partitioning. Two membrane fractions were obtained, one tentatively enriched with endoplasmic reticulum (ER) membranes and the other, with the plasma membrane (PM). The content of StHK2a and StHK4b in each fraction was determined by the radioligand method. Both receptors were detected in the two fractions, with a slight predominance of StHK4b in the ER-enriched fraction. Given that the content of ER membranes in the cell is markedly higher than the content of the PM, the bulk of the receptors should be attributed to the ER. Specific cytokinin-binding sites were detected in the analogous membrane fractions isolated from non-transformed potato roots. Although the specific content of the cytokinin-binding sites was 1.8-fold higher in the PM, here also, the main part of these sites, i.e., cellular receptors, still resided in the ER. In order to find out whether the receptors in the ER are functional, a recently proposed pH criterion was applied. According to this criterion, potato cytokinin receptors, like their orthologs from other species, seem to be tuned for functioning in the ER but not in the PM. The results of molecular modeling of the dimerization interface of the receptors at different pH values also evidenced in favor of the ER. Thus, the main site of subcellular localization of the functionally active potato cytokinin receptors is the ER, although a certain amount of the receptors resides within the PM, too. The question of the functional role of PM-located receptors remains open.