Abstract
Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.
Highlights
To determine which amino acids within the C terminus of DVMAT-A are necessary for internalization, we used site-directed mutagenesis to generate a series of C-terminal truncations (Fig. 1A)
Similar to our previously reported data [23], we found that wild-type DVMAT-A appeared to be completely internalized within 30 min at 23 °C (Fig. 2A)
We have investigated how potential trafficking domains in two structurally distinct vesicular transporters contribute to endocytosis in vitro and their localization to synaptic vesicles in vivo
Summary
Fly Stocks—Flies were raised on corn meal-molasses agar media at 23–25 °C and 20 – 40% humidity in a 12-h light/dark cycle. Western Blots—For Western blots comparing total dVMAT-A transgene expression in wt and mutant fly lines, flies were aged 3– 4 days, anesthetized using CO2, and either 6 or 7 heads per genotype were homogenized in buffer containing 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 1 mM EGTA, pH 8.0, 10% sucrose and proteinase inhibitor mixture (Roche Applied Science). For Western blot analysis of glycerol gradient fractionation, additional primary antibodies included an mAb directed against Drosophila cysteine string protein (DCSP, 1:2000, Developmental Studies Hybridoma Bank), which served as a marker for SVs [31, 32], rabbit anti-DVGLUT (1:5000) and rabbit anti-GFP (1:1000, Invitrogen, A-11122). The x-ray films representing the chemiluminescence signals from the input lanes and the gradient fractions containing SVs (the DCSP peak) were digitized and quantified for pixel intensity (ImageJ, NIH). Serial dilutions from each relevant fraction were quantified to improve precision
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