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 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.

• Publication year

  1997

• Venue

  Journal of Biological Chemistry

• Publication date

  1997-08-01

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1074/jbc.272.31.19165PMID 9235906
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• THI10 expression is regulated at the mRNA level by intracellular thiamin pyrophosphate and requires the positive regulator THI3.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• Expressing the THI10 open reading frame from the yeast GAL1 promoter restored both thiamin transport activity and thiamin binding activity in the disrupted cells.

  Confidence 0.98

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• Disrupting the THI10 locus eliminated both thiamin transport activity and thiamin binding activity in the yeast plasma membrane fraction.

  Confidence 0.99

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• THI10 was isolated from a Saccharomyces cerevisiae genomic library by complementation of a mutant defective in thiamin transport.

  Confidence 0.97

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review

• complementation

  method

  Functional rescue of a mutant phenotype by introducing a clone carrying the relevant gene.

  Aliases: complementation screening

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• gene disruption

  method

  Targeted inactivation of the THI10 locus in yeast cells.

  Aliases: disruption

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• genomic library

  method

  A collection of Saccharomyces cerevisiae genomic DNA clones used as the source for complementation screening.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• intracellular thiamin pyrophosphate

  metabolites

  The intracellular phosphorylated thiamin cofactor implicated in regulating THI10 transcript levels.

  Aliases: thiamin pyrophosphate, TPP

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• mrna-level regulation

  regulation processes

  Control of gene expression through changes in transcript abundance.

  Aliases: transcriptional/post-transcriptional mRNA control

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• plasma membrane fraction

  cell fractions

  The membrane-enriched yeast cell fraction assayed for thiamin transport and binding.

  Aliases: yeast plasma membrane fraction

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• thi10 gene

  gene

  A Saccharomyces cerevisiae gene identified here as the locus encoding the thiamin transporter candidate.

  Aliases: THI10, THI10 locus

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• thi3 gene

  regulatory genes

  A yeast positive regulatory gene required for THI10 expression in this study.

  Aliases: THI3

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• thiamin binding activity

  activity

  The plasma-membrane-associated ability to bind thiamin.

  Aliases: thiamine binding activity

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• thiamin transport activity

  activity

  The yeast plasma-membrane uptake activity that imports thiamin.

  Aliases: thiamine transport activity

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review
• yeast gal1 promoter

  promoters

  A galactose-inducible yeast promoter used to drive THI10 expression.

  Aliases: GAL1 promoter

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) review

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