title: Mitochondrial glutamate carriers from Drosophila melanogaster: biochemical, evolutionary and modeling studies.
abstract: The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.
authors.affiliation: Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Lunetti
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Cappello
authors.forename: René Massimiliano
authors.initials: RM
authors.lastname: Marsano
authors.forename: Ciro Leonardo
authors.initials: CL
authors.lastname: Pierri
authors.forename: Chiara
authors.initials: C
authors.lastname: Carrisi
authors.forename: Emanuela
authors.initials: E
authors.lastname: Martello
authors.forename: Corrado
authors.initials: C
authors.lastname: Caggese
authors.forename: Vincenza
authors.initials: V
authors.lastname: Dolce
authors.forename: Loredana
authors.initials: L
authors.lastname: Capobianco
mesh_terms: D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D019143:Evolution, Molecular; D005091:Exons / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007438:Introns / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D027322:Amino Acid Transport System X-AG; D017931:DNA Primers; D029721:Drosophila Proteins; C584606:GC1 protein, Drosophila; C584605:GC2 protein, Drosophila; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; C499216:SLC25A18 protein, human; C499215:SLC25A22 protein, human; D018698:Glutamic Acid
keywords: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-(N-morpholino)ethanesulfonic acid; CG18347 and CG12201; DmGC1; DmGC2; Drosophila melanogaster; Drosophila melanogaster glutamate carrier isoform 1; Drosophila melanogaster glutamate carrier isoform 2; GC; Glutamate carrier; HEPES; MES; NRG; Phylogenesis; Proteomics; SDS-PAGE; glutamate carrier; nuclear respiratory gene; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate