CLONING AND CHARACTERIZATION OF YEAST LEU4, ONE OF TWO GENES RESPONSIBLE FOR α-ISOPROPYLMALATE SYNTHESIS

Abstract

ABSTRACT By complementation of an α-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for α-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase Sal I-PvuII 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 LEU4fbr 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 α-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 α-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against α-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 LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized α-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second α-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.