Functional alcohol dehydrogenase mutants of Saccharomyces cerevisiae conferring temperature-conditional allyl alcohol...

Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature. A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.