Amino acid substitutions in membrane-spanning domains of Hol1, a member of the major facilitator superfamily of transporters, confer nonselective cation uptake in Saccharomyces cerevisiae.
Selection for the ability of Saccharomyces cerevisiae cells to take up histidinol, the biosynthetic precursor to histidine, results in dominant mutations at HOL1. The DNA sequence of HOL1 was determined, and it predicts a 65-kDa protein related to the major facilitator family (drug resistance subfamily) of putative transport proteins. Two classes of mutations were obtained: (i) those that altered the coding region of HOL1, conferring the ability to take up histidinol; and (ii) cis-acting mutations (selected in a mutant HOL1-1 background) that increased expression of the Hol1 protein. The ability to transport histidinol and other cations was conferred by single amino acid substitutions at any of three sites located within putative membrane-spanning domains of the transporter. These mutations resulted in the conversion of a small hydrophobic amino acid codon to a phenylalanine codon. Selection for spontaneous mutations that increase histidinol uptake by such HOL1 mutants resulted in mutations that abolish the putative start codon of a six-codon open reading frame located approximately 171 nucleotides downstream of the transcription initiation site and 213 nucleotides upstream of the coding region of HOL1. This single small upstream open reading frame (uORF) confers translational repression upon HOL1; genetic disruption of the putative start codon of the uORF results in a 5- to 10-fold increase in steady-state amounts of Hol1 protein without significantly affecting the level of HOL1 mRNA expression.
Wright
M B
MB
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Howell
E A
EA
Gaber
R F
RF
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent / Q000493:pharmacokinetics*; D004271:DNA, Fungal; D017353:Gene Deletion; D006641:Histidinol / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011188:Potassium / Q000378:metabolism; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic
D002352:Carrier Proteins; D002413:Cations, Divalent; D004271:DNA, Fungal; D008565:Membrane Proteins; D006641:Histidinol; D011188:Potassium