Phosphorylation-dependent dispersion of the response regulator in bacterial chemotaxis

Protein phosphorylation is a fundamental cellular regulatory mechanism that governs the activation and deactivation of numerous proteins. In two-component signaling transduction pathways, the phosphorylation of response regulator proteins and their subsequent diffusion play pivotal roles in signal transmission. However, the impact of protein phosphorylation on their dispersion properties remains elusive. In this study, using the response regulator CheY in bacterial chemotaxis as a model, we performed comprehensive measurements of the spatial distributions and diffusion characteristics of CheY and phosphorylated CheY through single-molecule tracking within live cells. We discovered that phosphorylation significantly enhances diffusion and mitigates the constraining influence of the cell membrane on these proteins. Moreover, we observed that ATP-dependent fluctuations also promote protein diffusion and reduce the restraining effect of the cell membrane. These findings highlight important effects of phosphorylation beyond protein activation.