Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae

We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

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GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.780 ◽

1987 ◽

Vol 7 (2) ◽

pp. 780-786 ◽

Cited By ~ 22

Author(s):

M I Riley ◽

J E Hopper ◽

S A Johnston ◽

R C Dickson

Keyword(s):

Saccharomyces Cerevisiae ◽

Type Strain ◽

Wild Type Strain ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Galactosidase Activity ◽

Wild Type ◽

Fold Induction ◽

Beta Galactosidase ◽

Galactokinase Activity

A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.

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Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.991-997.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 991-997

Author(s):

M Ruzzi ◽

K D Breunig ◽

A G Ficca ◽

C P Hollenberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Kluyveromyces Lactis ◽

Consensus Sequence ◽

Regulatory Gene ◽

Lac Operon ◽

Positive Regulation ◽

Strong Homology ◽

Gal Genes ◽

Beta Galactosidase ◽

Activation Site

In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.

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GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.780-786.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 780-786

Author(s):

M I Riley ◽

J E Hopper ◽

S A Johnston ◽

R C Dickson

Keyword(s):

Saccharomyces Cerevisiae ◽

Type Strain ◽

Wild Type Strain ◽

Glucose Repression ◽

Kluyveromyces Lactis ◽

Galactosidase Activity ◽

Wild Type ◽

Fold Induction ◽

Beta Galactosidase ◽

Galactokinase Activity

A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.

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Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.991 ◽

1987 ◽

Vol 7 (3) ◽

pp. 991-997 ◽

Cited By ~ 20

Author(s):

M Ruzzi ◽

K D Breunig ◽

A G Ficca ◽

C P Hollenberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Kluyveromyces Lactis ◽

Consensus Sequence ◽

Regulatory Gene ◽

Lac Operon ◽

Positive Regulation ◽

Strong Homology ◽

Gal Genes ◽

Beta Galactosidase ◽

Activation Site

In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.

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Evolution of moonlighting proteins: insight from yeasts

Biochemical Society Transactions ◽

10.1042/bst20140199 ◽

2014 ◽

Vol 42 (6) ◽

pp. 1715-1719 ◽

Cited By ~ 7

Author(s):

Carlos Gancedo ◽

Carmen-Lisset Flores ◽

Juana M. Gancedo

Keyword(s):

Saccharomyces Cerevisiae ◽

Present Article ◽

Catabolite Repression ◽

Kluyveromyces Lactis ◽

Approaches To Studying ◽

Moonlighting Proteins ◽

Experimental Approaches ◽

Gal Genes

The present article addresses the possibilities offered by yeasts to study the problem of the evolution of moonlighting proteins. It focuses on data available on hexokinase from Saccharomyces cerevisiae that moonlights in catabolite repression and on galactokinase from Kluyveromyces lactis that moonlights controlling the induction of the GAL genes. Possible experimental approaches to studying the evolution of moonlighting hexose kinases are suggested.

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Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1722 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1722-1730 ◽

Cited By ~ 21

Author(s):

G Cardinali ◽

V Vollenbroich ◽

M S Jeon ◽

A A de Graaf ◽

C P Hollenberg

Keyword(s):

Culture Medium ◽

Kluyveromyces Lactis ◽

Constitutive Expression ◽

Regulatory Function ◽

Nuclear Magnetic Resonance Analysis ◽

Resonance Analysis ◽

Crude Extracts ◽

Induction Process ◽

Beta Galactosidase ◽

Internal Production

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.

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Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures

Journal of Cell Science ◽

10.1242/jcs.61.1.339 ◽

1983 ◽

Vol 61 (1) ◽

pp. 339-349

Author(s):

J. Creanor ◽

S.G. Elliott ◽

Y.C. Bisset ◽

J.M. Mitchison

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Acid Phosphatase ◽

Kluyveromyces Lactis ◽

The Other ◽

Linear Pattern ◽

Synchronous Cultures ◽

Asynchronous Control ◽

Alpha Glucosidase ◽

Beta Galactosidase

Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S. cerevisiae and beta-galactosidase in Kluyveromyces lactis. There was no sign of step rises in activity in acid phosphatase but there were indications in S. cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S. pombe. There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques. Asynchronous control cultures showed little or no perturbations after the first hour.

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Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/125.2.281 ◽

1990 ◽

Vol 125 (2) ◽

pp. 281-291

Author(s):

P J Bhat ◽

D Oh ◽

J E Hopper

Keyword(s):

Saccharomyces Cerevisiae ◽

Signal Transduction Pathway ◽

Striking Similarity ◽

In Vitro Tests ◽

Multiple Copies ◽

Gal1 Gene ◽

Galactokinase Activity

Abstract The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.

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Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5427 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5427-5432 ◽

Cited By ~ 64

Author(s):

S J Smith ◽

J H Crowley ◽

L W Parks

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Control ◽

Membrane Integrity ◽

Fold Increase ◽

Regulatory Function ◽

Ergosterol Biosynthesis ◽

Galactosidase Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Yeast Genes ◽

Beta Galactosidase

Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae.

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