Mol Gen Genet (1986) 204:397~403 © Springer-Verlag1986 Yeast LEU5 is a PET-like gene that is not essential for leucine biosynthesis Peter Drain and Paul Schimmel Massachusetts Institute of Technology, Cambridge, MA 02139, USA Summary. Alpha-IPM synthase catalyzes the first commit- ted step in leucine biosynthesis in the yeast S. cerev&iae. LEU4 is known to encode this enzyme activity. A second gene, LEU5, has been proposed to encode a second enzyme with this activity. We cloned LEU5 and genetically defined the locus. LEU5 maps to chromosome VIII and is tightly linked to CEN8. Five different mutations in LEU5 were analyzed: a site- directed deletion and a disruption, as well as three distinct mutations produced by chemical mutagenesis. In a leu4 background, each leu5 mutation causes a Leu- phenotype; in a LEU4 background, none of the mutations alters the Leu+ phenotype. This shows that LEU5 is not essential for leucine biosynthesis. In either a leu4 or LEU4 back- ground, each Ieu5 mutation causes a glycerol- phenotype. This operationally defines LEU5 as a PET gene. Two distinct suppressors of the Pet- phenotype of leu5 strains have been isolated. These suppressors revert the Pet- phenotype of each of four mutant leu5 alleles that were tested. Suppression occurs regardless of the allele at LEU4. Moreover, the suppressors co-revert the Leu- phe- notype for each of the four leu5 mutations that is combined with a leu4 allele. This establishes the presence of a gene other than LEU5 that encodes a second alpha-IPM syn- thase. Further analysis provided no evidence for synthase activity that is encoded by LEU5. Key words: Amino acid biosynthesis -LEU4 - Glycerol Introduction Five genetic loci are assigned to leucine biosynthesis in the yeast Saccharomyces cerevisiae. LEU1 and LEU2 encode the second and third enzymes, respectively, in the pathway (Satyanarayana et al. 1968). The enzymes are located in the cytoplasm (Ryan et al. 1973). LEU3 encodes a positive factor necessary for induction of expression of LEU1 and LEU2 (Baichwal et al. 1984). LEU4 encodes alpha-IPM synthase (Baichwal et al. 1984) which catalyzes acetyl group transfer from acetyl-CoA to alpha-ketoisovaleric acid (the first committed step in leucine biosynthesis). This synthase is encoded by a nuclear gene (Baichwal et al. 1984; Chang Abbreviation." EMS, ethylmethane sulfonate; IPM, isopropylma- late; NPD, nonparental ditype; PD, parental ditype; TT, tetratype Offprint requests to: P. Schimmel et al. 1984, see below) and is localized to mitochondria (Ryan et al. 1973; Hampsey et al. 1983). Levels of alpha-IPM synthase respond to general amino acid control signals (Hsu et al. 1982) and the enzyme is subject to feedback inhibition by leucine (Satyanarayana et al. 1968; Ulm et al. 1972). Feedback inhibition is abol- ished in one class of LEU4 mutants which are resistant to normally toxic trifluoroleucine (Baichwal et al. 1984). A second class of mutations in LEU4 neither abolishes al- pha-IPM synthase activity nor creates a Leu- phenotype. The mutations of this class when coupled with a mutation in LEU5 cause a Leu-- phenotype and alpha-IPM synthase deficiency. It has been proposed, therefore, that two isoen- zymes are responsible for alpha-IPM synthase activity and that either LEU4 or LEU5 must be functional in order for that activity to be expressed. One suggestion is that LEU5 encodes an alpha-IPM synthase isozyme (Baichwal et al. 1984). We report here the cloning of LEU5 and the use of this clone to establish that LEU5 maps to the centromere of chromosome VIII. Gene disruption, gene deletion, and chemical mutagenesis of the locus show that LEU5 is opera- tionally a PET gene. Further investigation provided evi- dence for a genetic locus other than LEU5 that encodes a second alpha-IPM synthase activity. Materials and methods Transformation. Bacterial cells were transformed by the method of Dagert and Ehrlich (1979). Yeast cells were transformed by the method of Hinnen et al. (1978) or Ito et al. (1983). Strains, media, and genetics. Bacterial strains used were HBI01 (Boyer and Roulland-Dussoix 1969) and DB6507 (pyrF74::Tn5, isogenic to HBI01; from David Botstein, MIT). Yeast strains used are tabulated in Table 1. Bacterial and yeast media and general yeast genetic methods were as described (Davis et al. 1980; Sherman et al. 1979). The chromosomal assignment of LEU5 was determined by the benomyl-induced chromosomal loss method of Wood (1982). leu5 andpetl were distinguished in tetrad segregants by ability when combined with a leu4 mutation to result in a Leu- phenotype and by complementation testing of the Pet phenotype by crosses to known tester strains. D-, L-trifluoroleucine was purchased from Fairfield Chemical Co., Inc. (Blythewood, SC, U.S.A.), and used at 0.6 raM.