ISOLATION AND CHARACTERIZATION OF A THIAMIN TRANSPORT GENE, THI10, FROM SACCHAROMYCES-CEREVISIAE

We isolated a thiamin transporter gene, THI10, from a genomic library of Saccharomyces cerevisiae by the complementation of a yeast mutant d efective in thiamin transport activity, The THI10 gene contained an op en reading frame of 1,794 base pairs encoding a 598-amino acid polypep tide with a calculated molecular weight of 66,903, The nucleotide sequ ence of THI10 is completely identical to that of an anonymous DNA (ope n reading frame L8083.2) mapped to chromosome XII; two other genes (op en reading frames YOR071c and YOR192c) in chromosome XV are extremely similar to THI10, Moreover, the THI10 gene product showed significant sequence homology with yeast allantoin and uracil transporters, Hydrop athy profile suggested that THI10 product is highly hydrophobic and co ntains many transmembrane regions. Gene disruption of the THI10 locus completely abolished the thiamin transport activity and thiamin bindin g activity in yeast plasma membrane fraction, Both the transport and t hiamin binding activities were restored in the disrupted cells when th e THI10 open reading frame was expressed by yeast GAL1 promoter, sugge sting that the THI10 gene encodes for the thiamin transport carrier pr otein, Northern blot analysis demonstrated that THI10 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and that it requires a positive regulatory factor encoded by THI3 gene .