The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

Abstract

Synaptic vesicles (SV) are generated by two different mechanisms, one AP-2 dependent and one AP-3 dependent. It has been uncertain, however, whether these mechanisms generate SV that differ in molecular composition. We explored this hypothesis by analyzing the targeting of ZnT3 and synaptophysin both to PC12 synaptic-like microvesicles (SLMV) as well as SV isolated from wild-type and AP-3-deficient mocha brains. ZnT3 cytosolic tail interacted selectively with AP-3 in cell-free assays. Accordingly, pharmacological disruption of either AP-2-or AP-3-dependent SLMV biogenesis preferentially reduced synaptophysin or ZnT3 targeting, respectively; suggesting that these antigens were concentrated in different vesicles. As predicted, immuno-isolated SLMV revealed that ZnT3 and synaptophysin were enriched in different vesicle populations. Likewise, morphological and biochemical analyses in hippocampal neurons indicated that these two antigens were also present in distinct but overlapping domains. ZnT3 SV content was reduced in AP-3-deficient neurons, but synaptophysin was not altered in the AP-3 null background. Our evidence indicates that neuroendocrine cells assemble molecularly heterogeneous SV and suggests that this diversity could contribute to the functional variety of synapses. INTRODUCTIONThe molecular diversity in total brain synaptic vesicle (SV) composition is generally presumed to result from differential expression of synaptic vesicle membrane proteins in different brain regions. However, the possibility that synaptic vesicles differ in composition because of different biogenesis mechanisms has not been explored. Different vesiculation pathways could result in molecularly diverse synaptic vesicles. Vesiculation mechanisms are known to produce distinct cargo carriers from a population of donor membranes (Bonifacino and Dell'Angelica, 1999;Springer et al., 1999;Boehm and Bonifacino, 2001). This process is achieved by adaptor complexes that recognize and concentrate specific membrane proteins in the donor membranes (Bonifacino and Dell 'Angelica, 1999;Kirchhausen, 1999). PC12 cells have been shown to possess two such adaptor-dependent pathways for the assembly of synaptic vesicles, also known as synaptic-like microvesicles (SLMV) (Shi et al., 1998;de Wit et al., 1999;Jarousse and Kelly, 2001). In one pathway, SLMV are generated from the plasma membrane by a vesiculation mechanism that requires the adaptor AP-2, clathrin, and the GTPase dynamin (Shi et al., 1998). In the second pathway, SLMV are generated from endosomes by the adaptor complex AP-3 (Faundez et al., 1998;Blumstein et al., 2001) and the GTPase ARF1 (Faundez et al., 1997). In contrast to the plasma membrane pathway, the endosomederived mechanism is highly sensitive to brefeldin A (BFA) (Shi et al., 1998).Phenotypes observed in the AP-3-deficient mocha mouse are consistent with a role for the AP-3-dependent, endosome-derived pathway in neurons (Kantheti et al., 1998). The mocha mossy fibers are devoid of both the synaptic vesiclespecific zinc transpor...

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

“…Importantly, the content of synaptophysin in the ZnT3-containing fractions was substantially reduced after M␤CD treatment ( Figure 1B) without affecting ZnT3 content ( Figure 1B). As shown previously (Thiele et al, 2000;Salazar et al, 2004b), the M␤CD effects were not restricted to synaptophysin among SLMV proteins, suggesting that recovery of AP-2-generated SLMVs in this fraction is disrupted by plasma membrane cholesterol extraction.We further purified a ZnT3-enriched microvesicle fraction from the ZnT3-enriched peak of the glycerol gradient using isopycnic sedimentation. Fractions 8 -11 of the glycerol gradient were pooled and fractionated by equilibrium flotation in sucrose gradients (Supplemental Figure 1).…”

“…Immunoreactive bands were quantified using NIH Image 1.62 software as described previously (Salazar et al, 2004b).…”

“…Immunofluorescence was performed as described previously (Salazar et al, 2004b). All cells were seeded on coverslips coated with Matrigel (BD Bio-…”

“…Out-of-focus light was removed with a constrained iterative deconvolution algorithm (Swedlow et al, 1997). MetaMorph quantification was performed as described previously (Salazar et al, 2004b). Electron microscopy procedures are detailed in Salazar et al, 2004b.…”

“…MetaMorph quantification was performed as described previously (Salazar et al, 2004b). Electron microscopy procedures are detailed in Salazar et al, 2004b. Immunoperoxidase staining on brain sections has been described in detail previously (Salazar et al, 2004a).…”