CLONING AND CHARACTERIZATION O F YEAST LEU 4 , ONE O F T W O GENES RESPONSIBLE FOR a - ISOPROPYLMALATE SYNTHESIS

By complementation of an a-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu3), a plasmid was isolated that carried a structural gene for a-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase S a l I h I I segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU@‘ LEU5 strain, the resulting transformants had lost the 5’,5’,5’trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The a-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much a-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against a-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEUCspecific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized aisopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second a-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed. N yeast, as in other microorganisms and plants, leucine biosynthesis is accomI plished in four steps: (1) condensation of acetyl-coA and a-ketoisovalerate to a-isopropylmalate (a-IPM), catalyzed by a-IPM synthase; (2) isomerization of aIPM to p-IPM, catalyzed by IPM isomerase; (3) oxidative decarboxylation of pIPM to yield a-ketoisocaproate, catalyzed by p-IPM dehydrogenase; and (4) transamination of a-ketoisocaproate, catalyzed by branched-chain amino acid Abbreviations used: IPM, isopropylmalate; PMSF, phenylmethylsulfonyl fluoride; TPCK, tosylamino-8phenylethyl chloromethyl ketone; TLCK, tosyl-L-lysine chloromethyl ketone; YEPD. yeast extract-peptonedextrose; Kb, kilobasepairs. Genetics 10% 91-106 September, 1984. 92 L-F. L. CHANG ET AL. aminotransferase, Evidence presented previously suggests that IPM isomerase and 0-IPM dehydrogenase, which are encoded by LEU1 and LEU2, respectively, are induced by a-IPM, the product of the first reaction, in conjunction with a regulatory element produced by LEU3 (BAICHWAL et al. 1983). In this respect, leucine pathway regulation in S. cerevisiae is very similar to that described for another Ascomycete, viz., Neurospora crassa (for reviews see GROSS 1969; KOHLHAW 1983). Because of the likely involvement of a-IPM in controlling subsequent biosynthetic steps, a-IPM synthase would be expected to play a key role in leucine pathway regulation. This is indeed the case and is particularly obvious with yeast which appears to have evolved a rather sophisticated “a-IPM synthase system.” A recent genetic analysis showed that two genes, designated LEU4 and LEUS, must be mutated in order to generate an a-IPM synthase-negative phenotype (BAICHWAL et al. 1983). LEU4 was defined as a structural gene because an allele of LEU4 produces a feedback-resistant a-IPM synthase (BAICHWAL et al. 1983). The nature of LEU5 is uncertain, that is, it is not known whether it represents another structural gene or controls the expression of a structural gene. Available evidence suggests that yeast does contain a second a-IPM synthase activity that may account for up to 25% of the total synthase activity of wild-type cells (L. F. CHANG, P. R. GATZEK and G. B. KOHLHAW, unpublished results; see also DISCUSSION). The major a-IPM synthase activity is encoded by LEU4 (BAICHWAL et al. 1983) and, as will be shown in this paper, possesses properties very much resembling those of the yeast a-IPM synthase characterized in previous studies. Those studies demonstrated the following. (1) a-IPM synthase is inhibited by leucine (SATYANARAYANA, UMBARGER and LINDEGREN 1968; ULM, BOHME and KOHLHAW 1972). (2) The enzyme is subject to a highly specific inactivation by CoA (TRACY and KOHLHAW 1975, 1977). The CoA effect can be prevented or reversed by ATP and is viewed as a means that allows the yeast cell to channel acetyl-coA away from biosynthetic pathways and into the citric acid cycle whenever the acetyl-coA concentration reaches a certain low threshold value (HAMPSEY and KOHLHAW 1981). (3) The level of a-IPM synthase is regulated by the mechanism known as “general control” of amino acid biosynthesis (Hsu, KOHLHAW and NIEDERBERGER 1982). (4) a-IPM synthase consists of two apparently identical subunits whose molecular weight is 65,000-67,000 (TRACY and KOHLHAW 1977). (5) Much of the a-IPM synthase activity of yeast is found in the mitochondrial matrix (HAMPSEY, LEWIN and KOHLHAW 1983). Import into the mitochondria does not result in a measurable molecular weight change (HAMPSEY, LEWIN and KOHLHAW 1983; GASSER, DAUM and SCHATZ 1982). It is of interest from an evolutionary point of view that there are significant differences between the a-IPM synthase system of yeast and its Neurospora counterpart. The Neurospora enzyme, although inhibited by leucine, apparently is not inactivated by CoA and is localized in the cytosol; it also has much smaller subunits (molecular weight approximately 43,000) that aggregate to form trimers or tetramers (GROSS 1970; S. GROSS, personal communication). Expression of the Neurospora leu-4 gene, the gene that encodes a-IPM synthase, is negatively controlled by leucine and appears to be affected also by the leu-3 gene product (GROSS 1969). The present communication is part of an ongoing effort to identify and THE LEU4 GENEOF YEAST TABLE 1 Vectors and strains 93 Vectof or strain Description Source or reference YEpl3