Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae

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.     

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.     

Molecular characterization of MRG19 of Saccharomyces cerevisiae. Implication in the regulation of galactose and nonfermentable carbon source utilization.

We have reported previously that multiple copies of MRG19 suppress GAL genes in a wild-type but not in a gal80 strain of Saccharomyces cerevisiae. In this report we show that disruption of MRG19 leads to a decrease in GAL induction when S. cerevisiae is induced with 0.02% but not with 2.0% galactose. Disruption of MRG19 in a gal3 background (this strain shows long-term adaptation phenotype) further delays the GAL induction, supporting the notion that its function is important only under low inducing signals. As a corollary, disruption of MRG19 in a gal80 strain did not decrease the constitutive expression of GAL genes. These results suggest that MRG19 has a role in GAL regulation only when the induction signal is weak. Unlike the effect on GAL gene expression, disruption of MRG19 leads to de-repression of CYC1-driven beta-galactosidase activity. MRG19 disruptant also showed a twofold increase in the rate of oxygen uptake as compared with the wild-type strain. ADH2, CTA1, DLD1, and CYC7 promoters that are active during nonfermentative growth did not show any de-repression of beta-galactosidase activity in the MRG19 disruptant. Western blot analysis indicated that MRG19 is a glucose repressible gene and is expressed in galactose and glycerol plus lactate. Experiments using green fluorescent protein fusion constructs indicate that Mrg19p is localized in the nucleus consistent with the presence of a consensus nuclear localization signal sequence. Based on the above results, we propose that Mrg19p is a regulator of galactose and nonfermentable carbon utilization.     

Mutational hypersensitivity of a gene regulatory protein: Saccharomyces cerevisiae Gal80p

The inhibitor of galactose catabolic (GAL) gene expression in Saccharomyces cerevisiae, Gal80p, interacts with the activator Gal4p and the signal transducer Gal3p and self-associates. Selection for loss of Gal80p inhibitor function yielded gal80 mutants at an extremely high rate. Out of these, 21 nonoverlapping point mutants were identified; each were due to a single-amino-acid exchange in conserved residues. Semiquantitative biochemical analysis of the corresponding mutant proteins revealed that each of the 21 amino acid alterations caused simultaneous defects in every single protein-protein interaction and in Gal80's structural integrity. Thus, Gal80 provides an unprecedented example for a protein's structural sensitivity to minimal sequence alterations.     

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.     

Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4.

Temperature-sensitive (ts) mutants for the gal80 and gal4 genes of Saccharomyces cerevisiae were isolated and characterized. These mutants were classified into two categories; one showed thermolability (TL) and the other showed temperature-sensitive synthesis (TSS) of the respective products. Both the TL and TSS gal80 mutants are constitutive for galactokinase activity at 35 degrees C and, because they are derived from a dominant super-repressible GAL80s mutant, are uninducible at 25 degrees C. Both the TL and TSS gal4 mutants are galactose negative at 35 degrees C and galactose positive at 25 degrees C. None of the ts gal4 mutations affected the thermolability of galactokinase activity in cell extracts. Induction of galactokinase activity was studied with these mutants. The results indicate that the gal80 gene codes for a repressor and the gal4 gene codes for a positive factor indispensable for the expression of the structural genes or their products. However, striking evidence that the expression of the gal4 gene is constitutive and not under the control of gal80 was provided by a kinetic study with the TL gal4 mutant. The TL gal4 mutant pregrown in glycerol nutrient medium at 35 degrees C showed a prolonged lag period (35 min) in the induction of galactokinase activity at 25 degrees C, whereas the same mutant pregrown at 25 degrees C showed the same lag period as those observed in the wild-type strain and a revertant clone derived from the TL gal4 mutant (15 min).     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.     

Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.     

Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.