title: 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 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.
authors.affiliation: From the Department de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona.
authors.forename: Carla
authors.initials: C
authors.lastname: Serra-Peinado
authors.affiliation: From the Department de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona.
authors.forename: Adrià
authors.initials: A
authors.lastname: Sicart
authors.affiliation: the Facultad de Medicina de Albacete and Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, E-0200 Albacete, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Llopis
authors.affiliation: From the Department de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona, the Institut d'Investigació Biomèdica August Pi i Sunyer, E-08036 Barcelona, the Institut de Nanociència i Nanotecnologia (INUB), E-08036 Barcelona, and gegea@ub.edu.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Egea
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D025262:Vacuolar Proton-Translocating ATPases
keywords: Golgi; actin; intracellular trafficking; lipid raft; pH regulation; vacuolar ATPase