title: Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+transporter Gdt1p are required for calcium transport.
abstract: The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca2+transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca2+transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca2+-ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation.
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Colinet
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Louise
authors.initials: L
authors.lastname: Thines
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Antoine
authors.initials: A
authors.lastname: Deschamps
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Flémal
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Didier
authors.initials: D
authors.lastname: Demaegd
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
authors.forename: Pierre
authors.identifier: 0000-0001-7780-7230
authors.initials: P
authors.lastname: Morsomme
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D017920:Antiporters; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics; D027682:Cation Transport Proteins; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017920:Antiporters; D015220:Calcium Channels; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C575014:TMEM165 protein, human; C000614526:ribosome biosynthesis protein GDT1, S cerevisiae; D000252:Calcium-Transporting ATPases; D002118:Calcium
keywords: GDT1; Golgi; Saccharomyces cerevisiae; TMEM165; UPF0016; calcium transporter