Isolation and Characterization of a Thiamin Transport Gene,THI10, from Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 1997-08, Vol.272 (31), p.19165-19170
• Main Authors: Enjo, Fumio, Nosaka, Kazuto, Ogata, Masakazu, Iwashima, Akio, Nishimura, Hiroshi
• Format: Article
• Language: English
• Subjects: ACTIVE TRANSPORT; Amino Acid Sequence; AMINO ACID SEQUENCES; ARN MENSAJERO; ARN MESSAGER; Base Sequence; BINDING PROTEINS; Carrier Proteins - genetics; CELL MEMBRANES; CHEMICAL COMPOSITION; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; EXPRESION GENICA; EXPRESSION DES GENES; GENBANK/D55634; GENE; GENE EXPRESSION; Gene Expression Regulation; GENES; Genes, Fungal; GENETIC REGULATION; GENETICA; GENETICS; GENETIQUE; HIDROFOBICIDAD; HYDROPHOBICITE; HYDROPHOBICITY; INDUCED MUTATION; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; MESSENGER RNA; MOLECULAR MAPPING; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; MUTACION INDUCIDA; MUTANT; MUTANTES; MUTANTS; MUTATION PROVOQUEE; NUCLEOTIDE SEQUENCE; OPEN READING FRAMES; PESO MOLECULAR; PLASMA MEMBRANES; POIDS MOLECULAIRE; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; RESTRICTION MAPPING; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; STRUCTURAL GENES; THI10 GENE; THIAMIN; THIAMINE; Thiamine - metabolism; Thiamine Pyrophosphate - pharmacology; TIAMINA
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.31.19165

We isolated a thiamin transporter gene,THI10, from a genomic library of Saccharomyces cerevisiae by the complementation of a yeast mutant defective in thiamin transport activity. The THI10 gene contained an open reading frame of 1,794 base pairs encoding a 598-amino acid polypeptide with a calculated molecular weight of 66,903. The nucleotide sequence of THI10 is completely identical to that of an anonymous DNA (open reading frame L8083.2) mapped to chromosome XII; two other genes (open reading framesYOR071c and YOR192c) in chromosome XV are extremely similar to THI10. Moreover, the THI10gene product showed significant sequence homology with yeast allantoin and uracil transporters. Hydropathy profile suggested that THI10 product is highly hydrophobic and contains many transmembrane regions. Gene disruption of the THI10 locus completely abolished the thiamin transport activity and thiamin binding activity in yeast plasma membrane fraction. Both the transport and thiamin binding activities were restored in the disrupted cells when the THI10 open reading frame was expressed by yeast GAL1 promoter, suggesting that the THI10 gene encodes for the thiamin transport carrier protein. Northern blot analysis demonstrated thatTHI10 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and that it requires a positive regulatory factor encoded by THI3 gene.