Importance of phosphorylation/dephosphorylation cycles on lipid-dependent modulation of membrane protein topology by posttranslational phosphorylation

Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling pathways. Protein kinases and phosphatases have been described early on as key regulatory elements of the phosphorylated state of proteins. Tight spatial and temporal regulation of protein kinase and phosphatase activities has to be achieved in the cell to ensure accurate signal transduction. We demonstrated previously that phosphorylation of a membrane protein can lead to its topological rearrangement. Additionally, we found that both the rate and extent of topological rearrangement upon phosphorylation are lipid charge- and lipid environment-dependent. Here, using a model membrane protein (the bacterial lactose permease LacY reconstituted in proteoliposomes) and a combination of real-time measurements and steady-state assessments of protein topology, we established a set of experimental conditions to dissect the effects of phosphorylation and dephosphorylation of a membrane protein on its topological orientation. We also demonstrate that the phosphorylation-induced topological switch of a membrane protein can be reversed upon protein dephosphorylation, revealing a new regulatory role for phosphorylation/dephosphorylation cycles. Furthermore, we determined that the rate of topological rearrangement reversal is correlated with phosphatase activity and is influenced by the membrane's lipid composition, presenting new insights into the spatiotemporal control of the protein phosphorylation state. Together, our results highlight the importance of the compartmentalization of phosphorylation/dephosphorylation cycles in controlling membrane protein topology and, therefore, function, which are influenced by the local lipid environment of the membrane protein.