AP3M harbors actin filament binding activity that is crucial for vacuole morphology and stomatal closure in Arabidopsis.

Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole morphology are both involved in stomatal movement. The sorting of cargoes by clathrin adaptor protein (AP) complexes from the Golgi to the vacuole is critical for establishing a normal vacuole morphology. In this study, we demonstrate that the medium subunit of the AP3 complex (AP3M) binds to and severs actin filaments in vitro and that it participates in the sorting of cargoes (such as the sucrose exporter SUC4) to the tonoplast, and thereby regulates stomatal closure in Arabidopsis thaliana Defects in AP3 or SUC4 led to more rapid water loss and delayed stomatal closure, as well as hypersensitivity to drought stress. In ap3m mutants, the F-actin status was altered compared to the wild type, and the sorted cargoes failed to localize to the tonoplast. AP3M contains a previously unidentified F-actin binding domain that is conserved in AP3M homologs in both plants and animals. Mutations in the F-actin binding domain of AP3M abolished its F-actin binding activity in vitro, leading to an aberrant vacuole morphology and reduced levels of SUC4 on the tonoplast in guard cells. Our findings indicate that the F-actin binding activity of AP3M is required for the precise localization of AP3-dependent cargoes to the tonoplast and for the regulation of vacuole morphology in guard cells during stomatal closure.