title: Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter.
abstract: The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.
authors.affiliation: School of Biology, University of Saint Andrews, Fife, Scotland, UK. jrk@st-andrews.ac.uk
authors.forename: James R
authors.initials: JR
authors.lastname: Kinghorn
authors.forename: Joan
authors.initials: J
authors.lastname: Sloan
authors.forename: Ghassan J M
authors.initials: GJ
authors.lastname: Kana'n
authors.forename: Edisio R
authors.initials: ER
authors.lastname: Dasilva
authors.forename: Duncan A
authors.initials: DA
authors.lastname: Rouch
authors.forename: Shiela E
authors.initials: SE
authors.lastname: Unkles
mesh_terms: D000595:Amino Acid Sequence; D027321:Anion Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001234:Aspergillus niger / Q000235:genetics*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020125:Mutation, Missense; D000091285:Nitrate Transporters; D011487:Protein Conformation
chemical_list: D027321:Anion Transport Proteins; D005656:Fungal Proteins; D000091285:Nitrate Transporters