title: Vacuolar H(+)-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage.
abstract: The Vo sector of the vacuolar H(+)-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1-a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca(2+)-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed.
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, Ontario M5T 2S8, Canada.
authors.forename: Ner Mu Nar
authors.initials: NM
authors.lastname: Saw
authors.forename: Soo-Young Ann
authors.initials: SY
authors.lastname: Kang
authors.forename: Leon
authors.initials: L
authors.lastname: Parsaud
authors.forename: Gayoung Anna
authors.initials: GA
authors.lastname: Han
authors.forename: Tiandan
authors.initials: T
authors.lastname: Jiang
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Grzegorczyk
authors.forename: Michael
authors.initials: M
authors.lastname: Surkont
authors.forename: Ge-Hong
authors.initials: GH
authors.lastname: Sun-Wada
authors.forename: Yoh
authors.initials: Y
authors.lastname: Wada
authors.forename: Lijun
authors.initials: L
authors.lastname: Li
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D004298:Dopamine / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion; D009474:Neurons / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D020033:Protein Isoforms / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism; D015854:Up-Regulation; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D009478:Neuropeptide Y; D018377:Neurotransmitter Agents; D020033:Protein Isoforms; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D000469:Alkaline Phosphatase; D025262:Vacuolar Proton-Translocating ATPases; C569187:vacuolar ATPase V0 subunit A1, rat; C569188:vacuolar ATPase V0 subunit A2, rat; D004298:Dopamine; D009638:Norepinephrine