title: Vglut1 and ZnT3 co-targeting mechanisms regulate vesicular zinc stores in PC12 cells.
abstract: The lumenal ionic content of an organelle is determined by its complement of channels and transporters. These proteins reach their resident organelles by adaptor-dependent mechanisms. This concept is illustrated in AP-3 deficiencies, in which synaptic vesicle zinc is depleted because the synaptic-vesicle-specific zinc transporter 3 does not reach synaptic vesicles. However, whether zinc transporter 3 is the only membrane protein defining synaptic-vesicle zinc content remains unknown. To address this question, we examined whether zinc transporter 3 and the vesicular glutamate transporter Vglut1 (a transporter that coexists with zinc transporter 3 in brain nerve terminals) were co-targeted to synaptic-like microvesicle fractions in PC12 cells. Deconvolution microscopy and subcellular fractionation demonstrated that these two transporters were present on the same vesicles in PC12 cells. Vglut1 content in synaptic-like microvesicle fractions and brain synaptic vesicles was partially sensitive to pharmacological and genetic perturbation of AP-3 function. Whole-cell flow-cytometry analysis of PC12 cell lines expressing zinc transporter 3, Vglut1 or both showed that vesicular zinc uptake was increased by Vglut1 expression. Conversely, production of zinc transporter 3 increased the vesicular uptake of glutamate in a zinc-dependent fashion. Our results suggest that the coupling of zinc transporter 3 and Vglut1 transport mechanisms regulates neurotransmitter content in secretory vesicles.
authors.affiliation: Department of Cell Biology, Center for Neurodegenerative Disease, and Department of Pathology and Laboratory Medicine, Emory University, 615 Michael Street, Room 446, Atlanta, GA 30322, USA.
authors.forename: Gloria
authors.initials: G
authors.lastname: Salazar
authors.forename: Branch
authors.initials: B
authors.lastname: Craige
authors.forename: Rachal
authors.initials: R
authors.lastname: Love
authors.forename: Daniel
authors.initials: D
authors.lastname: Kalman
authors.forename: Victor
authors.initials: V
authors.lastname: Faundez
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D027682:Cation Transport Proteins / Q000378:metabolism / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D018698:Glutamic Acid / Q000737:chemistry; D007477:Ions; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D013569:Synapses / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050598:Vesicular Glutamate Transport Protein 1; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D007477:Ions; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C493466:Slc17a7 protein, rat; C491177:Slc30a3 protein, mouse; D050598:Vesicular Glutamate Transport Protein 1; C503697:zinc transporter 3, rat; D018698:Glutamic Acid; D015032:Zinc