Comparative proteomic profiling of receptor kinase signaling reveals key trafficking components enforcing plant stomatal development

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics remain underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass spectrometry, respectively. While ET-AP recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations in <i>PICALMs</i> impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Together, we provide a proteomic atlas of the ERECTA signaling network and demonstrate that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.