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

Yuanming Hu, Terrance Cooper, Gunter B. Kohlhaw

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 hinds to upstream activating sequences (UAS(LEU)) found in the promoters of LEU1, LEU2, LEU4, ILV2, and 1LV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of α-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when α-isopropylmalate is absent. Following identification of a UAS(LEU)-homologous sequence in the promoter of GDH1, the gene encoding NADP+-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UAS(LEU) 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 UAS(LEU) 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 α- isopropylmalate and is lowest in cells unable to produce α-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-α-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

Original languageEnglish (US)
Pages (from-to)52-57
Number of pages6
JournalMolecular and Cellular Biology
Volume15
Issue number1
DOIs
StatePublished - Jan 1 1995

Fingerprint

Saccharomyces cerevisiae Proteins
Glutamate Dehydrogenase (NADP+)
Nitrogen
Branched Chain Amino Acids
Null Lymphocytes
Biosynthetic Pathways
Genes
Lac Operon
Regulator Genes
Sequence Homology
Ammonia
Transcriptional Activation
Plasmids
Yeasts
Messenger RNA
Mutation
Enzymes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation. / Hu, Yuanming; Cooper, Terrance; Kohlhaw, Gunter B.

In: Molecular and Cellular Biology, Vol. 15, No. 1, 01.01.1995, p. 52-57.

Research output: Contribution to journalArticle

@article{eced1f7cf4b646b197ba2707f423796b,
title = "The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation",
abstract = "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 hinds to upstream activating sequences (UAS(LEU)) found in the promoters of LEU1, LEU2, LEU4, ILV2, and 1LV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of α-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when α-isopropylmalate is absent. Following identification of a UAS(LEU)-homologous sequence in the promoter of GDH1, the gene encoding NADP+-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UAS(LEU) 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 UAS(LEU) 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 α- isopropylmalate and is lowest in cells unable to produce α-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-α-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.",
author = "Yuanming Hu and Terrance Cooper and Kohlhaw, {Gunter B.}",
year = "1995",
month = "1",
day = "1",
doi = "10.1128/MCB.15.1.52",
language = "English (US)",
volume = "15",
pages = "52--57",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "1",

}

TY - JOUR

T1 - The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation

AU - Hu, Yuanming

AU - Cooper, Terrance

AU - Kohlhaw, Gunter B.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - 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 hinds to upstream activating sequences (UAS(LEU)) found in the promoters of LEU1, LEU2, LEU4, ILV2, and 1LV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of α-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when α-isopropylmalate is absent. Following identification of a UAS(LEU)-homologous sequence in the promoter of GDH1, the gene encoding NADP+-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UAS(LEU) 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 UAS(LEU) 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 α- isopropylmalate and is lowest in cells unable to produce α-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-α-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

AB - 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 hinds to upstream activating sequences (UAS(LEU)) found in the promoters of LEU1, LEU2, LEU4, ILV2, and 1LV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of α-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when α-isopropylmalate is absent. Following identification of a UAS(LEU)-homologous sequence in the promoter of GDH1, the gene encoding NADP+-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UAS(LEU) 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 UAS(LEU) 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 α- isopropylmalate and is lowest in cells unable to produce α-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-α-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

UR - http://www.scopus.com/inward/record.url?scp=0028936210&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028936210&partnerID=8YFLogxK

U2 - 10.1128/MCB.15.1.52

DO - 10.1128/MCB.15.1.52

M3 - Article

VL - 15

SP - 52

EP - 57

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 1

ER -