6533b872fe1ef96bd12d4423
RESEARCH PRODUCT
Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex*
Juan LlopisGustavo EgeaAdrià SicartCarla Serra-peinadosubject
0301 basic medicineVacuolar Proton-Translocating ATPasesGolgi ApparatusBiologyMicrofilamentBiochemistry03 medical and health sciencessymbols.namesakeCytosolHumansActin-binding proteinMolecular BiologyLipid raftActinGolgi membraneCell BiologyIntracellular MembranesGolgi apparatusHydrogen-Ion ConcentrationActin cytoskeletonCell biologyProtein Structure TertiaryCytosolActin Cytoskeleton030104 developmental biologysymbolsbiology.proteinHeLa Cellsdescription
We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. 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 the V1 domain, which in turn is coupled to the 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 depolymerization 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 alkalization. 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 membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.
year | journal | country | edition | language |
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2016-04-01 |