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First published online April 28, 2005
doi: 10.1242/10.1242/jcs.02319

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Vglut1 and ZnT3 co-targeting mechanisms regulate vesicular zinc stores in PC12 cells

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

View larger version (69K): [in a new window]   Fig. 1. Vglut1 and ZnT3 targeting to SLMVs are sensitive to brefeldin A. (A) PC12 cells transfected with Vglut1 and ZnT3 (VglZn clone 6 cells, VglZn6) were incubated in the absence or presence of 10 µg ml–1 BFA for 2 hours at 37°C. Cells were homogenized and equal protein amounts of S2 supernatant were resolved by glycerol-gradient sedimentation. Gradient fractions were analysed by immunoblot with antibodies against ZnT3, Vglut1 and synaptophysin (Sphysin). BFA decreased Vglut1 and ZnT3 targeting to SLMVs without affecting synaptophysin levels. (B) PC12 cells either expressing Vglut1 (Vgl cells) or co-expressing Vglut1 and ZnT3 (VglZn cells) were incubated in the absence or presence of BFA. Cell homogenates were fractionated and membrane fractions (P1, P2 and S2) were analysed by immunoblot with Vglut1 antibodies. After BFA treatment, Vglut1 decreases from the SLMV-enriched fraction (S2) independent of ZnT3 expression. The bracket on the left marks the migration of Vglut1, which characteristically migrates as a broad band (see Fig. 6B) (Bellocchio et al., 1998; Takamori et al., 2000a; Takamori et al., 2000b). Asterisk represents a cytosolic background band. (C) Untransfected cells (wt), PC12 clones expressing Vglut1 (Vgl) or Vglut1 and ZnT3 (VglZn) were fractionated by differential centrifugation and S2 enriched SLMV fractions were analysed by immunoblot using antibodies against the synaptic-vesicle markers SV2, synaptophysin (Sphysin) and VAMP II. Overexpression of transporters does not modify the SLMV content (n=3).

View larger version (69K): [in a new window]   Fig. 2. Vglut1 and ZnT3 colocalize in PC12 cells. PC12 cells expressing ZnT3 and Vglut1 were double labeled with antibodies against the HA tag present in ZnT3 and antibodies against Vglut1. Cell were imaged by wide-field deconvolution microscopy and colocalization was determined by Metamorph software using ten randomly picked cells. (A-C) Optical sections from the bottom of the cell to the top, spaced every 0.75 µm.

View larger version (57K): [in a new window]   Fig. 3. Vglut1 and ZnT3 are targeted to a common vesicle population. A glycerol gradient isolated SLMV fractions derived from Vglut1 and ZnT3HA (VglZn) double-transfected PC12 cells, and these were immunomagnetically isolated with beads coated with monoclonal antibodies against Vglut1 (A-C), antibodies against the HA tag present in ZnT3 (D, lane 2) or a monoclonal antibody against the AP-3 subunit (D, lane 3). Control assays were performed with beads coated with an antibody against a lumenal epitope of LAMP I. Bead-bound vesicles were resolved by SDS-PAGE and analysed by immunoblot with polyclonal antibodies against Vglut1, ZnT3 and the AP-3 ß3 subunit, and a monoclonal anti-transferrin-receptor (TrfR) antibody. (B) Unbound membranes were sedimented at high speed and analysed in parallel with bead-bound organelles depicted in (A,D). The Vglut1 blot was exposed for 30 seconds, whereas the TrfR blot was exposed overnight. Inputs correspond to 10%. Lanes 2 and 3 (A-C) represent duplicate assays.

View larger version (54K): [in a new window]   Fig. 4. Vglut1 and ZnT3 targeting to AP-3-deficient brain synaptic vesicles. (A) High-speed supernatants (S2) from wild-type (+/+) and mocha (–/–) brain homogenates were fractionated in 5-25% glycerol gradients to resolve small vesicles. Synaptic-vesicle antigen levels across gradients were determined by immunoblot using antibodies against synaptophysin (Sphysin), ZnT3 and Vglut1. ZnT3 and Vglut1 sedimentation patterns and the antigen contents of the membranes were altered in mocha brain vesicles. (B) The normalized content distribution of Vglut1 (n=3). No differences were found in the normalized distribution of synaptophysin (Kantheti et al., 1998; Salazar et al., 2004a). Closed circles represent wild-type membranes, open circles mocha vesicles. (C) The Vglu1 synaptic-vesicle level was determined in the peak fractions. The content of Vglut1 (n=3) was selectively reduced in mocha compared with control brain membranes. No changes were detected in the synaptophysin synaptic-vesicle levels (Kantheti et al., 1998; Salazar et al., 2004a). (D) S2 supernatants were obtained from wild-type (Ap3b2+/+) and neuronal AP-3-deficient brains (Ap3b2–/–) and fractionated as described in A. No appreciable differences were observed between brains lacking neuronal AP-3 and brains lacking both neuronal and ubiquitous AP-3 (n=3). ZnT3 synaptic-vesicle levels were partially affected in Ap3b2–/– (Seong et al., 2005).

View larger version (121K): [in a new window]   Fig. 5. Zinquin colocalizes with the synaptic-vesicle marker VAMP II in differentiated PC12 cells. (A) NGF-differentiated PC12 cells transfected with the early endosome marker syntaxin-13/GFP were zinc loaded, stained with zinquin and imaged by two-photon microscopy. Zinquin puncta and syntaxin-13/GFP failed to colocalize. (B) NGF-differentiated PC12 cells accumulate zinquin in vesicular compartments that (C) co-localize with the synaptic-vesicle marker VAMP-II/GFP.

View larger version (30K): [in a new window]   Fig. 6. Vglut1 increases the steady-state vesicular zinc content. Untransfected PC12 cells (wt), clones carrying either Vglut1 (Vgl), Vglut1 and ZnT3 (VglZn) or the VAMP II N49A mutant were zinc loaded and stained with zinquin. Zinquin fluorescence was determined by flow cytometry (A,C-E). (B) Immunoblot analysis of the expression levels of ZnT3 and Vglut1 in the cell types used in A,C-D. (A-C) Expression of Vglut1 (Vgl) increased zinquin fluorescence by 22%, comparing wild-type (wt) and Vgl cells. Zinquin fluorescence was further increased to 170% and 196% in two different clones co-expressing Vglut1 and ZnT3 (VglZn4 and VglZn6, respectively). No difference in zinquin fluorescence was observed in a PC12 clone carrying only ZnT3 (ZnT3-4). The graph depicts the average mean values of flow-cytometry profiles. Data were normalized with respect to untransfected PC12 cells (100%). `NS' is the background fluorescence determined in unstained cells. (D) Zinc loading was carried out in the absence or presence of bafilomycin A1 and zinc content was determined by zinquin fluorescence and flow cytometry. Bafilomycin A1 decreases zinquin fluorescence in Vgl and VglZn clones. Values are normalized to those obtained in the absence of drug (100%) for each cell type. Average ± s.e.m. Absent error bars are under the graphing threshold. (E) Representative zinquin flow-cytometry profile of untransfected PC12 cells (wt) and a PC12 clone carrying the VAMP II N49A mutant (n=7).

View larger version (17K): [in a new window]   Fig. 7. Zinc increases in vitro vesicular glutamate uptake. (A) SLMV-enriched fractions were prepared from untransfected (wt) and Vglut1-transfected (Vgl) cells. Vesicle-specific [3H]-glutamate uptake was determined in either the absence or the presence of the ionophore valinomycin (20 µM). Vesicular glutamate uptake is detected only in SLMV fractions from Vglut1-transfected cells (n=2). (B) [3H]-Glutamate uptake was performed in either the absence or the presence of increasing concentrations of ionic zinc (ZnSO4). Equal amounts of Vgl and VglZn6 SLMV protein were used in all assays. The protein content per assay was confirmed by immunoblot using antibodies against synaptic-vesicle markers.

View larger version (52K): [in a new window]   Fig. 8. Co-expression of zinc and glutamate transporters increases the steady-state vesicular glutamate content in whole cells. Untransfected PC12 cells (wt) or transfected either with Vglut1 (Vgl) or co-transfected with Vglut1 and ZnT3 (VglZn) were stained with glutamate (A,B,D,E) or Vglut1 antibodies (C). (A) Glutamate staining was mainly vesicular and was abolished by the BGG. Glutamate cellular content (B,D) increased in the presence of Vglut1 (Vgl) and co-expression of ZnT3 and Vglut1 further increased the cellular levels of glutamate (VglZn clones 4 and 6). NS, unstained cells; –Ab, cells stained only with the secondary antibody. (C) VglZn4 clonal cells produce less Vglut1 transporter than Vgl cells. (E) PC12 cells or clonal variants were treated in the absence or presence of bafilomycin A1 before antibody staining. Values are normalized to those obtained in the absence of drug (100%) for each cell type. (F) The vesicular index, representing vesicular glutamate pools, was 1.6 times greater in VglZn than in Vgl cells. Average ± s.e.m. Absent error bars are under the graphing threshold.