Vglut1 and ZnT3 co-targeting mechanisms regulate vesicular zinc stores in PC12 cells.
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.
Salazar
Gloria
G
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.
Craige
Branch
B
Love
Rachal
R
Kalman
Daniel
D
Faundez
Victor
V
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*
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