Novel role for an HPt domain in stabilizing the phosphorylated state of a response regulator domain

Two-component regulatory systems that utilize a multistep phosphorelay mech anism often involve a histidine-containing phosphotransfer (HPt) domain. Th ese HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domai n to another. In Saccharomyces cerevisiae, the YPD1 protein facilitates pho sphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct respons e regulator proteins, SSK1 and SKN7. Because the phosphorylation state larg ely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the thr ee response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively). The isolated regulatory domains exhibited phosphory lated lifetimes within the range previously observed for other response reg ulator domains (i.e., several minutes to several hours). However, in the pr esence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was d ramatically extended (almost 200-fold longer than in the absence of YPD1). This stabilization effect was specific for SSK1-R2, and was not observed fo r SLN1-R1 or SKN7-R3. Our findings suggest a mechanism by which SSK1 is mai ntained in its phosphorylated state under normal physiological conditions a nd demonstrate an unprecedented regulatory role for an HPt domain in a phos phorelay signaling system.