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

• Published in: The Journal of biological chemistry
• Main Authors: Serra-Peinado, Carla, Sicart, Adrià, Llopis, Juan, Egea Guri, Gustavo
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 12-02-2016
• Subjects: Aparell de Golgi; Citosquelet; Cytoskeletal proteins; Cytoskeleton; Enzimologia; Enzymology; Golgi apparatus; Homeostasis; Homeòstasi; Proteïnes citosquelètiques
• ISSN: 0021-9258

We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.