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.
Highlights
Membrane proteins present in a vesicle define the lumenal content of small-molecule mediators such as ions and amino acids
In order to analyse whether ZnT3 transport activity could be under control of the glutamate transporter Vglut1, we first determined whether ZnT3 and Vglut1 were segregated to the same vesicle population
Clonal PC12 cells doubly transfected with Vglut1 and ZnT3 (VglZn cells) were treated with or without brefeldin A (BFA) for 2 hours at 37°C (Fig. 1)
Summary
Membrane proteins present in a vesicle define the lumenal content of small-molecule mediators such as ions and amino acids. This is important in secretory vesicles, like synaptic vesicles, in which the lumenal contents act as neurotransmitters or neuromodulators that determine targetcell responses. The mocha allele results in a lack of AP-3 and is characterized by a profound neurological phenotype (Kantheti et al, 1998; Kantheti et al, 2003). We have determined that the mocha neurological phenotype emerges in part from defects in the assembly of synaptic vesicles. Synaptic-vesicle zinc stores are severely reduced from AP3–/– brain synapses (Kantheti et al, 1998; Kantheti et al, 2003)
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