Membrane topology of the Drosophila vesicular glutamate transporter

Department of Psychiatry and Biobehavioral Sciences, The David Geffen School of Medicine at UCLA, Gonda (Goldschmied) Center for Genetic and Neuroscience Research, Los Angeles, CA 90095-1761, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 06/2007; 101(6):1662-71. DOI: 10.1111/j.1471-4159.2007.04518.x
Source: PubMed


The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. Here we present data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, we have determined the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein. Using immunoisolation, we have identified complementary tagged sites that face the cytoplasm. Our data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins.

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Available from: Hao Fei, Sep 24, 2014
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    • "Vesicular glutamate transporters are fundamental components in glutamate signaling and are evolutionarily old. A vesicular glutamate transporter has been characterized in the tunicate Ciona intestinalis [22] as well as in Drosophila melanogaster [23] and Caenorhabditis elegans [24]. Also the presence of SLC17A9 in many animal species has been suggested [8]. "
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    • "VGLUT1 and VGLUT2 have a complementary distribution in rodent adult brain (Fremeau et al., 2001; Herzog et al., 2001; Hallberg et al., 2006). Membrane topology analysis of VGLUT2 revealed potential sites of phosphorylation by PKC and casein kinase II, which could regulate transport activity and trafficking (Jung et al., 2006; Fei et al., 2007). Glutamate-induced increase in acidification was detected in synaptic vesicles (Maycox et al., 1988) and in microvesicles from bovine pineal glands (Moriyama and Yamamoto, 1995), suggesting that an increase in VGLUT transport activity would cause an increase in ΔpH. "
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    ABSTRACT: The driving force for neurotransmitter accumulation into synaptic vesicles is provided by the generation of a transmembrane electrochemical gradient (DeltamicroH+) that has two components: a chemical gradient (DeltapH, inside acidic) and an electrical potential across the vesicular membrane (DeltaPsi, inside positive). This gradient is generated in situ by the electrogenic vacuolar H(+)-ATPase, which is responsible for the acidification and positive membrane potential of the vesicle lumen. Here, we investigate the modulation of vesicle acidification by using the acidic-organelle probe LysoTracker and the pH-sensitive probe LysoSensor at goldfish Mb-type bipolar cell terminals. Since phosphorylation can modulate secretory granule acidification in neuroendocrine cells, we investigated if drugs that affect protein kinases modulate LysoTracker staining of bipolar cell terminals. We find that protein kinase C (PKC) activation induces an increase in LysoTracker-fluorescence. By contrast, protein kinase A (PKA) or calcium/calmodulin kinase II (CaMKII) activation or inhibition did not change LysoTracker-fluorescence. Using a pH-dependent fluorescent dye (LysoSensor) we show that the PKC activation with PMA induces an increase in LysoSensor-fluorescence, whereas the inactive analog 4alpha-PMA was unable to cause the same effect. This increase induced by PMA was blocked by PKC inhibitors, calphostin C and staurosporine. These results suggest that phosphorylation by PKC may increase synaptic vesicle acidification in retinal bipolar cells and therefore has the potential to modulate glutamate concentrations inside synaptic vesicles.
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