Localization of the Ethylene-Receptor Signaling Complex to the Endoplasmic Reticulum: Analysis by Two-Phase...

Ethylene receptors and other elements of the ethylene-signal transduction pathway localize to membranes of the endoplasmic reticulum (ER). New players in the ethylene signaling pathway continue to be discovered and so it is important to have methods by which to diagnose their cellular localization. Two methods for microsome isolation and fractionation are described here that can assist in determining if a protein localizes to the ER: aqueous two-phase partitioning and equilibrium density-gradient centrifugation. Two-phase partitioning serves to purify plasma membrane away from other cellular membranes and can thus discriminate whether a protein is localized to the plasma membrane or not. Equilibrium density-gradient centrifugation is particularly useful for resolving if a protein is localized to the ER. Ribosomes are associated with the rough ER in the presence of Mg²⁺ but are stripped away when Mg²⁺is removed from the medium, resulting in a reduction in the ER membrane density and a diagnostic shift in migration when analyzed by equilibrium density-gradient centrifugation. A method for growing plants in liquid culture is also provided because these microsomal membranes exhibit consistent fractionation by both two-phase partitioning and density-gradient centrifugation.