Actin filaments are involved in the coupling of V0-V1 domains of vacuolar H+-ATPase at the Golgi complex

Abstract We previously reported that actin-depolymerizing agents promote the alkalinisation of the Golgi stack and the trans-Golgi network (TGN). The main determinant of acidic pH at the Golgi is the Vacuolar-type H+-translocating ATPase (V-ATPase), whose V1 domain subunits B and C bind actin. We have generated a GFP-tagged subunit B2 construct (GFP-B2) that is incorporated into V1 domain, which in turn is coupled with V0 sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation and inversal FRAP experiments show that the actin depolymerisation promotes the dissociation of V1-V0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by D-ceramide-C6 causes Golgi pH alkalinisation. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of Detergent Resistant Membranes (DRMs) organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.