Title: Molecular and functional characterization of SLC17A5 and SLC17A9 transporters in Drosophila melanogaster
Other Titles: Moleculaire en functionele karakterisering van SLC17A5 en SLC17A9 transporters in Drosophila melanogaster
Authors: Laridon, Bram
Issue Date: 5-Apr-2012
Abstract: Transporters are of major importance to living organisms. They function as gatekeepers for cells and organelles by controlling the uptake and efflux of various compounds and are therefore essential for an organism to maintain homeostasis. It is generally estimated that at least 5% of all human genes code for transporters, which further underscores their biological importance.The solute carrier (SLC) family of transporters represents the second largest family of membrane proteins (after the G-protein-coupled receptors) and contains in humans at least 378 proteins, divided into 51 SLC families. Among these, the SLC17 family mediates the transmembrane transport of organic anions. Proteins of the SLC17 family are involved in urate secretion (SLC17A1 and SLC17A3), lysosomal export of sialic acid (SLC17A5), and vesicular storage of aspartate (SLC17A5), glutamate (SLC17A5-8) and nucleotides (SLC17A9). Defects in each of these processes can have profound consequences and lead to diseases, underscoring the importance of proper function of these transporters.Despite the important biological functions of SLC17 transporters, studies have mainly been confined to the vesicular glutamate transporters SLC17A6-8 (VGLUT1-3). The other SLC17 transporters have received very little attention and most of the available data are based on in vitro experiments. Using the genetically tractable model organism Drosophila melanogaster we aim to gain further mechanistic insight into the function of SLC17 transporters. Specifically, we focused on the functional and genetic characterization of SLC17A5 (Sialin/VEAT) and SLC17A9 (VNUT) homologs in D. melanogaster.SLC17A5 (Sialin/VEAT) functions as a lysosomal H+/sialic acid cotransporter (Sialin) and mediates the export of free sialic acid out of the lysosome. When present in synaptic vesicles, SLC17A5 functions as a vesicular excitatory amino acid transporter (VEAT) and mediates membrane potential-dependent uptake of aspartate and glutamate. Mutations in SLC17A5 underlie the allelic lysosomal storage diseases Salla disease and infantile sialic acid storage disease (ISSD), characterized by severe neurological problems and an intralysosomal accumulation of sialic acid.In this study, we identified a group of Drosophila homologs of SLC17A5 (Sialin/VEAT) and studied their expression patterns throughout embryogenesis. We show that Drosophila SLC17A5-related genes are expressed in a variety of tissues, of which the uncharacterized genes CG3036 and CG8098 are expressed in the embryonic nervous system. We next performed a molecular-genetic and functional characterization of CG3036, which we renamed nestor. We show that Nestor displays SLC17A5-like transport properties and its expression is enriched in neurosecretory cells, where it colocalizes with the corresponding neuropeptides. Loss of Nestor does not cause a lysosomal storage disease, but increases lifespan up to 100%, in the absenceof an effect on fecundity or physical activity. Extension of lifespan and healthspan in nestor mutants is accompanied by increased resistance to starvation and oxidative stress. We provide evidence that Nestor acts in multiple tissues to control lifespan and starvation resistance and show that Nestor acts in insulin-producing cells to control resistance to oxidative stress. We hypothesize that the extension of lifespan and increased stress resistance of nestor mutants arises, at least partially, from a neuroendocrine disorder.Molecular-genetic and functional analysis of Drosophila CG8098, which we renamed pygmy, reveals that both loss of Pygmy and ubiquitous overexpression cause substantial growth retardation and lethality. In addition, we find that nervous system- specific overexpression causes excessive fluid retention and impaired defecation and is associated with a bloated phenotype, suggestive of impaired leucokinin signaling.Taken together, our study of the Drosophila SLC17A5 homologs Nestor and Pygmy points towards a role for SLC17A5-like transporters in neuropeptide secretion. This finding might well be relevant for sialic acid storage diseases and awaits further investigation.SLC17A9 represents the vesicular nucleotide transporter (VNUT), responsible for the uptake of ATP into synaptic vesicles, a process crucial to purinergic neurotransmission. We report the identification of the uncharacterized Drosophila gene CG15438 as the Drosophila vesicular nucleotide transporter (dVNUT). We show that dVNUT is a membrane potential- dependent ATP transporter, which potentially also transports GTP, UTP, ADP and AMP. We demonstrate that the ATP transport activity of dVNUT is strongly dependent on Cl- and can be inhibited by DIDS and Evans Blue, two known inhibitors of mammalian VNUT. dVNUT expression is strongly enriched in embryonic, larval and adult salivary glands and is also upregulated in the adult nervous system, ovary and male accessory glands. Our loss-of- function analysis reveals that dVNUT is not an essential gene in Drosophila. Although additional studies are needed to reveal the function of dVNUT, our data suggest a possible role for purinergic neurotransmission in the salivary glands and the nervous system in Drosophila.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Behavioral and Developmental Genetics
Department of Human Genetics - miscellaneous

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