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 domin ant mutations at HOL1, The DNA sequence of HOL1 was determined, and it predicts a 65-kDa protein related to the major facilitator family (dr ug resistance subfamily) of putative transport proteins, Two classes o f mutations were obtained: (i) those that altered the coding region of HOL1, conferring the ability to take up histidinol; and (ii) cis-acti ng mutations (selected in a mutant HOL1-1 background) that increased e xpression of the Hell 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 t he transporter, These mutations resulted in the conversion of a small hydrophobic amino acid codon to a phenylalanine codon, Selection for s pontaneous mutations that increase histidinol uptake by such HOL1 muta nts resulted in mutations that abolish the putative start codon of a s ix-codon open reading frame located approximately 171 nucleotides down stream of the transcription initiation site and 213 nucleotides upstre am of the coding region of HOL1, This single small upstream open readi ng frame (uORF) confers translational repression upon HOL1; genetic di sruption of the putative start codon of the uORF results in a 5- to 10 -fold increase in steady-state amounts of Hell protein without signifi cantly affecting the level of HOL1 mRNA expression.