We will explore three potential mechanisms by which the ethylene receptors may regulate activity of CTR1. First, we will test if the ethylene receptors increase CTR1 activity through a stable mechanism, such as the promotion of autophosphorylation. If this is the outcome, we will then screen for phosphorylated residue(s) on CTR1 from this system using mass spectrometry (MS) analysis. These could result from receptor induced autophosphorylation of CTR1, or perhaps ERS1/ETR1 may phosphorylate CTR1 as they have been shown to have Ser/Thr kinase activity. We will compare the phosphorylation of CTR1 purified from yeast in the presence and absence of the receptors by MS analysis. Previous studies have indicated multiple autophosphorylation sites on CTR1, primarily within the activation loop and the kinase P-loop and these and/or other residues may be activated in vivo through interaction with the receptors.…show more content… A third mechanism is that the receptors simply act to localize CTR1 to the ER membrane, juxtaposing it to EIN2, where it is then capable of phosphorylating EIN2. These two latter mechanisms will be distinguished by comparing the relative phosphorylation of EIN2 in the presence of soluble CTR1 and CTR1 tethered to the membrane via fusion to the N-terminal transmembrane domain of the ethylene receptor alone (i.e. a receptor lacking the cytosolic domain that normally facilitates the interaction with CTR1). This would be similar to the activation of RAF kinase, which is activated by localization to the plasma membrane via interaction with activated RAS, a GTP-binding protein that activates