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G B Kohlhaw

Publications and source records attributed to G B Kohlhaw.

At least 19 recordsLinked to original sources
PubMed1999

Yeast transcriptional regulator Leu3p. Self-masking, specificity of masking, and evidence for regulation by the intracellular level of Leu3p.

Recent work suggests that the masking of the activation domain (AD) of yeast transactivator Leu3p, observed in the absence of the metabolic signal alpha-isopropylmalate, is an intramolecular event. Much of the evidence came from the construction and analysis of a mutant form of Leu3p (Leu3-dd) whose AD is permanently masked (Wang, D., Hu, Y., Zheng, F., Zhou, K., and Kohlhaw, G. B. (1997) J. Biol. Chem. 272, 19383-19392). In a modified two-hybrid experiment, the ADs of both wild type Leu3p and Leu3-dd were shown to interact with the remainder of the Leu3 protein, in an alpha-isopropylmalate-dependent manner. The finding that masking and unmasking proceed apparently normally when full-length Leu3p is expressed in mammalian cells is also consistent with the notion of intramolecular masking. Here we report on the identification of nine missense mutations (all of them suppressors of the Leu3-dd phenotype) that cause permanent unmasking of Leu3p. The nine mutations map to three short segments located within a 140-residue-long region of the C-terminal part of the middle region of Leu3p. These segments may be part of a spatial trap for the AD. We also performed "domain swaps" between Leu3p and Cha4p, a serine/threonine-responsive activator that, like Leu3p, belongs to the family of Zn(II)2Cys6 proteins. We show that AD masking and response to the appropriate metabolic signal only occur when a given AD remains attached to its own middle region; middle region swapping results in constitutively active proteins. Finally, we show that the extent to which Leu3p regulates reporter gene expression depends on the intracellular concentration of Leu3p. The possible physiological significance of this observation is discussed in light of the known regulation of Leu3p by Gcn4p.

Amino Acid Substitution↗
PubMed1997

Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p.

The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of alpha-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191-195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for alpha-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking.

Amino Acid Sequence↗
PubMed1996

Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer.

The Leu3 protein of yeast is a dual-function regulator, stimulating transcription when the inducer alpha-isopropylmalate (alpha-IPM) is present and suppressing transcription when the inducer is absent. Here we show that Leu3p retains both its positive and negative regulatory properties when expressed in mammalian cells or when added to a mammalian nuclear extract. Alpha-IPM stimulates reporter gene expression 15-20-fold, both in vivo and in vitro. The concentration of alpha-IPM required for half-maximal stimulation in vitro is 2.5 x 10(-4) M. No yeast-specific factors other than Leu3p itself are required for up- or down-regulation. Since alpha-IPM is not metabolized in mammalian cells, the Leu3p-alpha-IPM system might be useful in gene therapy and other studies as a highly specific, externally controlled on/off switch of gene expression.

Animals↗
PubMed1995

Additive activation of yeast LEU4 transcription by multiple cis elements.

The LEU4 gene of Saccharomyces cerevisiae and the enzyme encoded by LEU4, alpha-isopropylmalate synthase, occupy a special position in amino acid metabolism. alpha-Isopropylmalate synthase catalyzes the first committed step in leucine biosynthesis. However, the reaction product alpha-isopropylmalate is not only an intermediate in the leucine biosynthetic pathway, but also functions as co-activator of at least six genes, both within and outside of the leucine pathway. The metabolic importance of alpha-isopropylmalate appears to be reflected in the surprisingly multifaceted regulation of LEU4 expression. This report describes an analysis of functional cis elements in the LEU4 promoter. Five such elements were identified. Three distal elements, designated UASLEU, GCE-A, and GCE-B, are responsible for regulation by the regulatory proteins Leu3p and Gen4p, respectively. The incremental activation of LEU4 by these elements is additive and independent. In addition, two proximal elements were localized. One of these conforms to the TATA consensus sequence and exhibits high affinity for TATA binding protein. The other element shows strong sequence identity with the Bas2p binding site and appears to be involved in basal and phosphate-mediated regulation of LEU4.

2-Isopropylmalate Synthase↗
PubMed1995

The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

Base Sequence↗
PubMed1994

Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.

The Leu3 protein (Leu3p) of Saccharomyces cerevisiae is a pleiotropic transregulator that can function both as an activator and as a repressor of transcription. It binds to upstream promoter elements (UASLEU) with the consensus sequence 5'-GCCGGNNCCGGC-3'. The DNA-binding motif of Leu3p belongs to the family of Zn(II)2-Cys6 clusters. The motif is located between amino acid residues 37 and 67 of the 886-residue protein. In this study, we used a recombinant peptide consisting of residues 17 to 147 to explore the interaction between Leu3p and its cognate DNA. We found that the Leu3p(17-147) peptide is a monomer in the absence of UASLEU but assumes a dimeric structure when the DNA is present. Results of protein-DNA cross-linking and methylation and ethylation interference footprinting experiments show that the Leu3p(17-147) dimer interacts symmetrically with two contact triplets separated by 6 bp and suggest that the peptide approaches its target DNA in such a way that each subunit is positioned closer to one DNA strand than to the other. The binding of Leu3p is strongly affected by the spacing between the contact triplets of the UASLEU and by the type of triplet. Binding occurs when the triplets are 6 bp apart (normal spacing) but fails to occur when the triplets are 0, 5, or 8 bp apart. Weak binding occurs when the triplets are 7 bp apart. Binding does not occur when the UASLEU triplets (GCC....GGC) are replaced with triplets found in the UAS elements for Gal4p, Put3p, and Ppr1p (CGG....CCG). The apparent Kd for the normal Leu3p(17-147)-UASLEU complex is about 3 nM. A mutant form of Leu3p(17-147) in which the histidine at position 50 has been replaced with cysteine binds UASLEU with significantly greater affinity (apparent Kd of about 0.7 nM), even though the interaction between the mutant peptide and target DNA appears to be unchanged. Interestingly, repression of basal-level transcription, which is a hallmark property of the wild-type Leu3p(17-147) peptide, is largely lost with the mutant peptide, indicating that there is no direct correlation between strength of binding and repression.

Binding Sites↗
PubMed1993

Purification and structural characterization of transcriptional regulator Leu3 of yeast.

The transcriptional regulatory protein Leu3 of Saccharomyces cerevisiae was enriched approximately 70-fold above wild type level in yeast cells carrying a pGAL1-LEU3 expression vector. Sustained overproduction of Leu3 following induction by galactose required elevated intracellular levels of alpha-isopropylmalate, a leucine pathway intermediate known to act as transcriptional co-activator. Starting with galactose-induced cells, the Leu3 protein was purified about 3,500-fold (i.e. 245,000-fold over wild type level) by a procedure that included treatment of the cell-free extract with polyethylenimine, fractionation with ammonium sulfate, heat treatment, and DNA affinity chromatography. Highly purified preparations still showed two protein bands when subjected to polyacrylamide electrophoresis under denaturing conditions. Their apparent molecular masses were about 104,000 and 110,000 kDa. The smaller of these values was very close to the maximum molecular weight obtained previously for Leu3 protein translated in vitro in a rabbit reticulocyte lysate. (The molecular weight deduced from the open reading frame of the LEU3 gene is 100,162.) Both protein bands reacted with antibodies raised against different portions of the Leu3 molecule and were, therefore, likely to represent two forms of Leu3. Treatment with calf intestinal phosphatase quantitatively converted the slower moving band into the faster moving one. Conversion was prevented by inorganic phosphate, a phosphatase inhibitor. These experiments showed that the two bands very likely correspond to phosphorylated and nonphosphorylated forms of Leu3. Phosphorylation did not appear to affect the DNA binding function of Leu3, but (indirect) effects on the activation function or effects on the modulation by alpha-isopropylmalate have not been ruled out. Electrophoretic mobility shift assays were used to estimate the apparent dissociation constants of the two specific Leu3-DNA complexes routinely seen in these assays. The values obtained were 1.1 and 2.6 nM. Finally, using size exclusion chromatography, native Leu3 protein was shown to have dimeric structure, irrespective of the state of phosphorylation.

Base Sequence↗
PubMed1993

Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

Animals↗
PubMed1992

In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate.

In the absence of the leucine biosynthetic precursor alpha-isopropylmalate (alpha-IPM), the yeast LEU3 protein (Leu3p) binds DNA and acts as a transcriptional repressor in an in vitro extract. Addition of alpha-IPM resulted in a dramatic increase in Leu3p-dependent transcription. The presence of alpha-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4/VP16, for limiting transcription factors. Therefore, the addition of alpha-IPM appears to convert a transcriptional repressor into an activator. This represents an example in eukaryotes of direct transcriptional regulation by a small effector molecule.

Binding, Competitive↗
PubMed1991

Manipulation of the 'zinc cluster' region of transcriptional activator LEU3 by site-directed mutagenesis.

The transcriptional activator LEU3 of Saccharomyces cerevisiae belongs to a family of lower eukaryotic DNA binding proteins with a well-conserved DNA binding motif known as the Zn(II)2Cys6 binuclear cluster. We have constructed mutations in LEU3 that affect either one of the conserved cysteines (Cys47) or one of several amino acids located within a variable subregion of the DNA binding motif. LEU3 proteins with a mutation at Cys47 were very poor activators which could not be rescued by supplying Zn(II) to the growth medium. Mutations within the variable subregion were generally well-tolerated. Only two of seven mutations in this region generated poor activators, and both could be reactivated by Zn(II) supplements. Three of the other five mutations gave rise to activators that were better than wild type. One of these, His50Cys, exhibited a 1.5 fold increase in in vivo target gene activation and a notable increase in the affinity for target DNA. The properties of the His50Cys mutant are discussed in terms of a variant structure of the DNA binding motif. During the course of this work, evidence was obtained suggesting that only one of the two LEU3 protein-DNA complexes routinely seen actually activates transcription. The other (which may contain an additional protein factor) does not.

Amino Acid Sequence↗
PubMed1990

Transcriptional activator LEU3 of yeast. Mapping of the transcriptional activation function and significance of activation domain tryptophans.

The LEU3 protein of yeast activates a number of genes in the branched chained amino acid pathways. Native LEU3 is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. alpha-Isopropylmalate is needed for transcriptional activation, but not for DNA binding. We show here that the transcriptional activation function of LEU3 resides within the C-terminal 32 amino acids. An adjacent stretch of 81 residues is dispensable and apparently forms a connecting link between the activation domain and a large central region previously identified as important for modulation. The newly defined activation domain contains a cluster of three tryptophan residues, each of which was changed to alanine by site-directed mutagenesis. Surprisingly, all three Trp----Ala mutations affect modulation. One of them, Trp-864----Ala, creates a LEU3 molecule that is largely unmodulated and also is a better transcriptional activator than is wild type LEU3 ("hyperactivator"). The other two mutations (Trp-861----Ala and Trp-870----Ala) change the modulation ratio but have no effect on the maximal activation efficiency of the activator. We propose that the activation domain of LEU3 is kept silent by association with the central region of the protein and that an alpha-isopropylmalate-induced conformational change in the central region releases and thus activates the activation domain.

Base Sequence↗
PubMed1990

Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2.

We have constructed a total deletion of the regulatory gene LEU3. Comparing the deletion mutant with a leu3 spontaneous mutant, we find that both types of mutants have lost the ability to regulate a LEU2'-lacZ translational fusion by the LEU3-alpha-isopropylmalate-dependent mechanism, which we confirm to be the major regulatory mechanism for LEU2. Surprisingly, cells containing the total leu3 deletion are more leaky (i.e. grow better in the absence of extraneous leucine) than cells containing a spontaneous leu3 mutation. Accompanying the growth rate difference is a difference in the expression of the LEU2-lacZ fusion: the specific activity of beta-galactosidase amounts to about 8% of a wild type control in a leu3 total deletion mutant, but drops to about 2% in a leu3 spontaneous mutant. The spontaneous mutant differs from the total deletion mutant in that it produces an inactive protein which is still able to bind to the LEU2 upstream activating sequence. We conclude that a basal level control of LEU2 becomes manifest in the absence of LEU3 and is interfered with when LEU3 protein binds to the LEU2 promoter. This conclusion is supported by the finding that a mutant which contains an intact LEU3 gene but is unable to generate alpha-isopropylmalate also interferes with basal level expression of LEU2. Basal level expression depends upon the GCN4 protein, even though LEU2 is not subject to derepression by the general amino acid control system. Changes in the steady-state concentration of LEU2 mRNA show the same trend as changes in the specific activity of the LEU2-lacZ fusion protein, suggesting that regulation of LEU2 expression at both the basal and nonbasal levels is largely transcriptional. The role of alpha-isopropylmalate in the regulation of LEU2 expression appears to be that of a co-activator. Employing mobility shift assays, we show that specific interaction between the LEU3 protein and a 30-base pair DNA fragment carrying the upstream activating sequence of LEU2 takes place irrespective of the presence or absence of alpha-isopropylmalate.

2-Isopropylmalate Synthase↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Expression of the yeast LEU4 gene is subject to four different modes of control.

A translational fusion of yeast LEU4 and Escherichia coli lacZ which contains 679 bp of the LEU4 5'-flanking region and the first two codons of LEU4 was used to study LEU4 expression. Eight recipient strains with different genetic backgrounds, transformed with a plasmid containing the fusion, were grown under a variety of conditions, and beta-galactosidase activity was measured. Evidence was obtained for at least four modes of expression of LEU4: general amino acid control, leucine-specific control, basal level expression, and branched-chain amino acid-mediated repression. Determination of steady-state levels of LEU4 mRNA suggested that LEU4 expression is regulated transcriptionally.

2-Isopropylmalate Synthase↗
PubMed1988

Yeast LEU4 encodes mitochondrial and nonmitochondrial forms of alpha-isopropylmalate synthase.

The LEU4 gene of Saccharomyces cerevisiae is the major structural gene involved in the production of alpha-isopropylmalate synthase. It was recently proposed that LEU4 should be capable of encoding two forms of alpha-isopropylmalate synthase, based mainly on the observation that two of four major transcription start sites are located downstream from the ATG at the beginning of the LEU4 open reading frame (Beltzer, J. P., Chang, L. L., Hinkkanen, A. E., and Kohlhaw, G. B. (1986) J. Biol. Chem. 261, 5160-5167). The two forms with molecular weights of 68,000 and 65,000, respectively, would differ only in the N-terminal region, and only the larger of the two forms would be imported into the mitochondria. We have now constructed LEU4'-'lacZ translational fusion plasmids that contain either a normal LEU4' portion (expected to express both the long and the short forms of the fusion protein) or a modified LEU4' portion in which productive translation is possible only from the second in-frame AUG (expected to express only the short form of the fusion protein). beta-Galactosidase measurements and immunoblotting of crude mitochondrial and cytoplasmic fractions of yeast cells transformed with the fusion plasmids indicate that two forms of hybrid protein are produced and that only the larger form is targeted to the mitochondria. The distinguishing feature of the targeting sequence appears to be its ability to form an 18-residue long amphiphilic helix. Expression of the native short form of LEU4-encoded alpha-isopropylmalate synthase behind a strong yeast promoter has enabled us to show that the short form is functional in leucine biosynthesis, is inhibited by leucine with an apparent inhibitor constant of approximately 0.4 mM, and exists as a cytoplasmic dimer.

2-Isopropylmalate Synthase↗