Identification and characterization of the thiamine transporter gene of Saccharomyces cerevisiae.
A positive selection scheme is described that selects for thiamine transporter clones. The scheme is based on the rescue of lethality, under non-permissive conditions, of Saccharomyces cerevisiae strains that are conditional for thiamine biosynthesis and are defective in thiamine transport. Transport defective strains were generated by selection for resistance to the lethal thiamine analog, pyrithiamine. Pyrithiamine resistance was shown to be a recessive, single gene trait that resulted from the mutation of the thiamine transporter gene, as suggested by previous work. Conditional thiamine biosynthesis was generated by cloning THI4, a thiamine biosynthetic gene, into a URA3 containing plasmid and transforming a strain disrupted in THI4. Thus, plating on 5-fluoroorotic acid causes the loss of thiamine synthesis ability. The gene for the yeast thiamine transporter, THI7, was cloned using this scheme. The predicted 598 amino acid transporter is a member of the major facilitator superfamily of transporters and thus possesses 12 transmembrane spanning segments with amino and carboxy termini intracellularly located. Several alterations in the coding region were characterized that result in greatly reduced ability to transport thiamine. The level of transporter mRNA was found to be rapidly and dramatically reduced by the addition of thiamine to the growth medium.
Singleton
C K
CK
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA. Singleck@ctrvax.Vanderbilt.edu
D000135:Acid Phosphatase; D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004352:Drug Resistance, Microbial / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011745:Pyrithiamine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013831:Thiamine / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; C000592466:Thi7 protein, S cerevisiae; C026989:thiamine-binding protein; D011745:Pyrithiamine; D000135:Acid Phosphatase; C488913:PHO3 protein, S cerevisiae; C488916:PHO5 protein, S cerevisiae; D013831:Thiamine