Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-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 alpha-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 alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-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 alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.     

Asp578 in LEU4p is one of the key residues for leucine feedback inhibition release in sake yeast

We identified a new mutation, Asp578Tyr, in alpha-isopropylmalate synthase (a LEU4 gene product) that releases leucine feedback inhibition and causes hyperproduction of isoamyl alcohol (i-AmOH) in sake yeast. Spontaneous sake yeast mutants that express resistance to 5,5,5-trifluoro-DL-leucine (TFL) were isolated, and a mutant strain, TFL20, was characterized at the genetic and biochemical levels. An enzyme assay for alpha-isopropylmalate synthase showed that strain TFL20 was released from feedback inhibition by L-leucine. Furthermore, DNA sequencing of the LEU4 gene for a haploid of the mutant TFL20 revealed that aspartic acid in position 578 changes to tyrosine. A comparison of the three-dimensional structures of wild-type LEU4p and mutant LEU4D578Yp by the homology modeling method showed that Asp578 is important for leucine feedback inhibition. We conclude that the mutation from Asp to Tyr in 578 is a novel change causing release from leucine feedback inhibition.     

Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage

Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV.     

Yeast regulatory protein LEU3: a structure-function analysis.

Eleven mutations resulting in partially deleted or truncated LEU3 protein were generated by linker insertion or other modifications at restriction sites, deletion of restriction fragments, or oligonucleotide-directed mutagenesis. Functional studies of these mutants showed the following: (i) A specific DNA binding region is contained within the 173 N-terminal residues, but other regions of the protein are required for optimal binding. (ii) Activation of LEU2 expression depends on the C-terminal 113 residues of the LEU3 protein. (iii) Deletion of part or all of a central section of LEU3 eliminates the ability of the LEU3 protein to respond to the co-activator alpha-isopropylmalate, i.e. creates an unmodulated activator. (iv) Overproduction of unmodulated activator slows down cell growth. (v) Specific deletion of two short acidic regions, including one with net charge - 19, has only minor effects on activation and modulation.     

Biosynthesis of branched-chain amino acids in yeast: effect of carbon source on leucine biosynthetic enzymes.

The three enzymes in the leucine biosynthetic pathway of yeast do not exhibit coordinate repression and derepression in response to the carbon source available in the culture medium. Growth in an acetate medium results in derepression of the first enzyme in the pathway, alpha-isopropylmalate synthase, and repression of the second two enzymes, alpha-isopropylmalate isomerase and beta-isopropylmalate dehydrogenase, relative to the levels found in glucose-grown cells. The role of endogenous leucine pools as a mediator of these differences was investigated. The leucine pools did not differ significantly between acetate-grown and glucose-grown cells. However, an elevated endogenous leucine pool, caused by exogenous leucine in the growth medium, did decrease the rate of decay of alpha-isopropylmalate synthase activity observed when acetate-grown cells were shifted to glucose. Evidence is provided suggesting that an elevated endogenous leucine pool may increase the in vivo stability of alpha-isopropylmalate synthase under several different conditions. Studies on the kinetics of alpha-isopropylmalate synthase decay in vivo and sensitivity to leucine inhibition indicate that there are two classes of the enzyme in acetate-grown yeast cells.     

Biosynthesis of branched-chain amino acids in yeast: regulation of leucine biosynthesis in prototrophic and leucine auxotrophic strains

The first enzyme in the biosynthesis of leucine in yeast, alpha-isopropylmalate synthetase, is inhibited by l-leucine. In a mutant resistant to the analogue 5',5',5'-trifluoroleucine, the enzyme is markedly resistant to inhibition by l-leucine. Growth ing the presence of exogenous l-leucine results in repression of the second and third enzymes of the pathway. The first enzyme is not repressed unless both l-leucine and l-threonine are supplied in the medium. Comparison of levels of the remaining two enzymes in leucine auxotrophs grown under conditions of leucine excess and leucine limitation reveals deviations from the wild-type derepression pattern in some mutants. In some, repression of the synthetase by leucine alone was observed. In others, the repressibility of the dehydrogenase was lost. It is unlikely that these deviations were due to the same primary mutational event that caused leucine auxotrophy. No mutants were found in which an altered gene was recognized to be clearly responsible for the level of the leucine-forming enzymes.     

Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product

It has been known that enzyme activity associated with the yeast LEU1 and LEU2 gene product (beta-isopropylmalate dehydrogenase) drops sharply when yeast is grown in the presence of leucine. RNA blot hybridizations with LEU2-specific probes establish that this is accompanied by a 5-fold repression in LEU2 mRNA levels. A similar repression was noted recently for LEU1 mRNA levels (Hsu, Y.-P., and Schimmel, P. (1984) J. Biol. Chem. 259, 3714-3719). Nuclease mapping of the 5'-end of the LEU2 mRNA shows a major start at approximately 16 nucleotides upstream of the AUG initiation codon. This initiation site in the gene is retained in an extensive LEU2 5'-noncoding region deletion which still expresses the LEU2 gene product (Erhart, E., and Hollenberg, C. P. (1983) J. Bacteriol. 156, 625-635). The primary structure of the LEU2 gene product was established from the nucleotide sequence of the gene-coding region and from fitting amino acid sequences of scattered internal peptides to the nucleotide sequence. The 364-amino acid protein has a 13-amino acid stretch which is highly homologous to the partially sequenced yeast LEU1 gene product (isopropylmalate isomerase). The homology occurs about 290 amino acids from the respective NH2 termini of the two proteins. The homology may represent residues which interact with beta-isopropylmalate, a common ligand for the enzymes.     

Comparative genomics of transcriptional regulation in yeasts and its application to identification of a candidate alpha-isopropylmalate transporter

Conservation rates in non-protein-coding regions of five yeast genomes of the genus Saccharomyces were analyzed using multiple whole-genome alignments. This analysis confirmed previously shown decrease in conservation rates observed immediately upstream of the translation start point and downstream of the stop-codon. Further, there was a sharp conservation peak in the upstream regions likely related to the core promoter (-35 bp to +35 bp around TSS) and a conservation peak downstream of the stop-codon whose function is not yet clear. Regulation of leucine and methionine biosynthesis controlled by the global regulator Gcn4p and pathway-specific regulators was analyzed in detail. A candidate alpha-isopropylmalate carrier, YOR271cp, was identified based on conservation of Leu3p binding sites, analysis of ChIP-chip data, protein localization and sequence similarity.