In Arabidopsis, stomatal patterning is directed by receptor complexes involving the ERECTA-family (ERf) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins. The endosomal sorting complex required for transport (ESCRT) facilitates endocytic degradation of membrane proteins, while its specific role in modulating stomatal patterning remains elusive.

Here, we show that the ESCRT-III-associated proteins VPS46.1 and VPS46.2 function redundantly to govern stomatal patterning. Loss of VPS46 function leads to excessive, disorganized stomatal lineage divisions and clustering. Genetic analyses position VPS46 downstream of EPF2 and upstream of the YODA-Mitogen-Activated Protein Kinase (MAPK) cascade.

VPS46 proteins localized to late endosomes and colocalized with ERf receptors. The VPS46 mutation specifically disrupted the vacuolar degradation of the ERf-SERKs complex, terminally trapping the receptors on the tonoplast and halting their further cycling. By contrast, the trafficking and function of the brassinosteroid receptor BRI1 were unaffected.

Our study identifies VPS46 as a critical regulator that determines the postendocytic fate of the ERf-SERKs receptor complex. It reveals a novel substrate-selective mechanism within the ESCRT pathway, whereby VPS46 ensures the precise spatial patterning of stomata by facilitating degradation of the ERf-SERKs complex to fine-tune signaling output.