Purification and characterization of the yeast negative regulatory protein GAL80

Sang Jung Yun, Yoshiki Hiraoka, Masafumi Nishizawa, Koji Takio, Koiti Titani, Yasuhisa Nogi, Toshio Fukasawa

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80% was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.

Original languageEnglish
Pages (from-to)693-697
Number of pages5
JournalJournal of Biological Chemistry
Volume266
Issue number2
Publication statusPublished - 1991 Jan 15

Fingerprint

Yeast
Purification
Yeasts
Galactose
Proteins
Transcription
DEAE-Cellulose Chromatography
Ammonium Sulfate
Streptomycin
Mutant Proteins
DEAE-Cellulose
Column chromatography
Gene encoding
Gel Chromatography
Saccharomyces cerevisiae
Immune Sera
Metabolism
Alleles
Amino Acids
Monomers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Yun, S. J., Hiraoka, Y., Nishizawa, M., Takio, K., Titani, K., Nogi, Y., & Fukasawa, T. (1991). Purification and characterization of the yeast negative regulatory protein GAL80. Journal of Biological Chemistry, 266(2), 693-697.

Purification and characterization of the yeast negative regulatory protein GAL80. / Yun, Sang Jung; Hiraoka, Yoshiki; Nishizawa, Masafumi; Takio, Koji; Titani, Koiti; Nogi, Yasuhisa; Fukasawa, Toshio.

In: Journal of Biological Chemistry, Vol. 266, No. 2, 15.01.1991, p. 693-697.

Research output: Contribution to journalArticle

Yun, SJ, Hiraoka, Y, Nishizawa, M, Takio, K, Titani, K, Nogi, Y & Fukasawa, T 1991, 'Purification and characterization of the yeast negative regulatory protein GAL80', Journal of Biological Chemistry, vol. 266, no. 2, pp. 693-697.
Yun SJ, Hiraoka Y, Nishizawa M, Takio K, Titani K, Nogi Y et al. Purification and characterization of the yeast negative regulatory protein GAL80. Journal of Biological Chemistry. 1991 Jan 15;266(2):693-697.
Yun, Sang Jung ; Hiraoka, Yoshiki ; Nishizawa, Masafumi ; Takio, Koji ; Titani, Koiti ; Nogi, Yasuhisa ; Fukasawa, Toshio. / Purification and characterization of the yeast negative regulatory protein GAL80. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 2. pp. 693-697.
@article{7430f3882b0c4525a7d4cf7801ec38b6,
title = "Purification and characterization of the yeast negative regulatory protein GAL80",
abstract = "Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80{\%} was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.",
author = "Yun, {Sang Jung} and Yoshiki Hiraoka and Masafumi Nishizawa and Koji Takio and Koiti Titani and Yasuhisa Nogi and Toshio Fukasawa",
year = "1991",
month = "1",
day = "15",
language = "English",
volume = "266",
pages = "693--697",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "2",

}

TY - JOUR

T1 - Purification and characterization of the yeast negative regulatory protein GAL80

AU - Yun, Sang Jung

AU - Hiraoka, Yoshiki

AU - Nishizawa, Masafumi

AU - Takio, Koji

AU - Titani, Koiti

AU - Nogi, Yasuhisa

AU - Fukasawa, Toshio

PY - 1991/1/15

Y1 - 1991/1/15

N2 - Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80% was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.

AB - Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80% was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.

UR - http://www.scopus.com/inward/record.url?scp=0026033043&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026033043&partnerID=8YFLogxK

M3 - Article

VL - 266

SP - 693

EP - 697

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -