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Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p. 总被引:4,自引:3,他引:1 下载免费PDF全文
T Suzuki-Fujimoto M Fukuma K I Yano H Sakurai A Vonika S A Johnston T Fukasawa 《Molecular and cellular biology》1996,16(5):2504-2508
The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids. 相似文献
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Genetic and Molecular Characterization of Gal83: Its Interaction and Similarities with Other Genes Involved in Glucose Repression in Saccharomyces Cerevisiae 总被引:9,自引:2,他引:7 下载免费PDF全文
Expression of the GAL genes of Saccharomyces cerevisiae is subject to glucose repression, a global regulatory mechanism that requires several gene products. We have isolated GAL83, one of these genes required for glucose repression. The sequence of the predicted Gal83 protein is homologous to two other yeast proteins, Sip1p and Sip2p, which are known to interact with the SNF1 gene product, a protein kinase required for expression of the GAL genes. High-copy clones of SIP1 and SIP2 cross-complement the GAL83-2000 mutation (as well as GAL82-1, a mutation in another gene involved in glucose repression), suggesting that these four genes may perform similar functions in glucose repression. Consistent with this hypothesis, a gal83 null mutation does not affect glucose repression, and only dominant or partially dominant mutations exist in GAL83 (and GAL82). Two other observations were made that suggests that GAL83 functions interdependently with GAL82 and REG1 (another gene involved in glucose repression) to effect glucose repression: 1) REG1 on a low-copy plasmid cross-complements GAL82-1 and GAL83-2000 mutations, and 2) all pairwise combinations of reg1, GAL82-1 and GAL83-2000 fail to complement one another. Such unlinked noncomplementation suggests that Gal83p, Gal82p and Reg1p may interact with one another. Possible roles for GAL83, GAL82 and REG1 are discussed in relation to SNF1, SIP1 and SIP2. 相似文献
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Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae 总被引:12,自引:2,他引:10 下载免费PDF全文
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. 相似文献