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Volume 272, Number 31, Issue of August 1, 1997 pp. 19165-19170
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, February 18, 1997, and in revised form, April 18, 1997)

From the Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, and the ^§ Kyoto Prefectural Institute of Hygienic and Environmental Science, Fushimi-ku, Kyoto 612, Japan

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 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. 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 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.

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