SummaryUnderstanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem. A multitude of genes in different pathways are repressed by glucose at the level of transcription. The SUC2 gene, which encodes invertase, is an excellent reporter gene for glucose repression, since its expression is controlled exclusively by this pathway. Genetic analysis has identified numerous regulatory mutations which can either prevent derepression of SUC2 or render its expression insensitive to glucose repression. These mutations allow us to sketch the outlines of a pathway for general glucose repression, which has several key elements: hexokinase Pll, encoded by HXK2, which seems to play a role in the sensing of glucose levels; the protein kjnase encoded by SNF1, whose activity is required for derepression of many glucose-repressible genes; and the MIG1 repressor protein, which binds to the upstream regions of SUC2 and other glucoserepressible genes. Repression by MIG1 requires the activity of the CYC8 and TUP1 proteins. Glucose repression of other sets of genes seems to be controlled by the general glucose repression pathway acting in concert with other mechanisms. In the cases of the GAL genes and possibly CYC1, regulation is mediated by a cascade in which the general pathway represses expression of a positive transcriptional activator.

The TUPI and CYC8 (=SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUPI or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MATa cells. The TUP) gene was isolated in a screen for genes that regulate mating type (V. L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tupl-100. (21,34,44,49).tupi and cyc8 mutants share many phenotypes, including calcium-dependent flocculation (34,36,50), mating-type defects in MATa cells (5,20,34,54), nonsporulation of * Corresponding author.homozygous diploids (34, 36), and derepression of many enzymes under glucose repression. The diversity of phenotypes may arise from the involvement of TUPI and CYC8 in regulatory pathways distinct from glucose repression or may reflect the interaction of glucose repression with these pathways.SNFJ (CCRI, CATJ) encodes a protein kinase that is necessary for the derepression of glucose-repressible genes (6, 9). Mutant snfl strains cannot ferment sucrose or utilize other nonfermentable carbon sources. Some of these effects are suppressed by cyc8 mutations. Double snfl cyc8 mutants are constitutive for invertase synthesis (6). It has been postulated that CYC8 is a negative regulator of glucose repression and that SNFJ may act through CYC8 (6).Mutations in TUPI and CYC8 produce the same array of phenotypes, suggesting that the two genes function at similar points in regulation. However, their specific roles and the mechanism of glucose repression are still unknown, although the latter seems to involve multiple steps or pathways (11,27). Possible roles for TUPI and CYC8 as negative regulators could be repression of transcription or interference of activation of transcription. Recently CYC8 (SSN6) was cloned, characterized, and sequenced (37, 51). However, no functional role has yet been identified for the CYC8 gene product. To further understand the roles of TUP1 and CYC8 in catabolite repression, TUPI has been characterized and sequenced. This work is described here. MATERIALS AND METHODSStrains and media. The Saccharomyces cerevisiae strains used (Table 1)

Mutations of the yeast CYC8 or TUPI genes greatly reduce the degree of glucose repression of many genes and affect other regulatory pathways, including mating type. The predicted CYC8 protein contains 10 copies of the 34-amino-acid tetratricopeptide repeat unit, and the predicted TUP1 protein has six repeated regions found in the I8 subunit of heterotrineric G proteins. The absence of DNA-binding motifs and the presence of these repeated domains suggest that the CYC8 and TUP1 proteins function via protein-protein interaction with transcriptional regulatory proteins. We raised polyclonal antibodies against TrpE-CYC8 and TrpE-TUPl fusion proteins expressed in Escherichia coli. The CYC8 and TUP1 proteins from yeast cells were detected as closely spaced doublets on Western immunoblots of sodium dodecyl sulfate-polyacrylamide gels. Western blots of nondenaturing gels revealed that both proteins are associated in a high-molecular-weight complex with an apparent size of 1,200 kDa. In extracts from Acyc8 strains, the size of the complex is reduced to 830 kDa. The CYC8 and TUP1 proteins were coprecipitated by either antiserum, further supporting the conclusion that they are associated with each other. The complex could be reconstituted in vitro by mixing extracts from strains with complementary mutations in the CYC8 and TUPI genes.Carbon catabolite repression is a widespread phenomenon among microorganisms whereby the synthesis of enzymes required for the utilization of alternate carbon sources is inhibited in the presence of the preferred carbon source. In the yeast Saccharomyces cerevisiae, glucose or fructose are the preferred carbon sources and the process is usually referred to as glucose repression. Yeast cells grown in the presence of glucose repress the synthesis of many classes of enzymes, including those required for metabolism of other carbon sources, enzymes involved in gluconeogenesis and respiration, and vacuolar hydrolases such as proteases. In all cases which have been examined, regulation occurs at the level of transcription.Our laboratory isolated mutations in two genes, tupi and cyc8, which abolish glucose repression of SUC2, which encodes invertase (37). Mutations in the tupi and cyc8 genes had been isolated previously for their effects on phenotypes other than glucose repression. The tupi (thymidine uptake) mutants were first isolated for their ability to take up dTMP from the growth medium (41). Mutations in the same gene were subsequently isolated and given various names according to the phenotype of interest: umr7,flkl, amml, and cyc9.The umr7 mutants were resistant to UV-induced mutation of CAN] to cani (19,20). flkl mutants were extremely flocculent or "flaky" and were insensitive to catabolite repression of maltase, invertase, and a-methylglucosidase (28, 35). amml mutants stabilized plasmids containing a defective ARS element (36). A selection protocol for increased expression of iso-2-cytochrome c yielded cyc9, which is allelic to tupi, and a new mutant, cyc8 (24). Mutations in CYC8 were late...

The Cyc8 (Ssn6)-Tup1 corepressor complex is required for repression in several important regulatory systems in yeast cells, including glucose repression and mating type. Cyc8-Tup1 is recruited to target genes by interaction with diverse repressor proteins that bind directly to DNA. Since the complex has a large apparent molecular mass of 1,200 kDa on nondenaturing gels (F. E. Williams, U. Varanasi, and R. J. Trumbly, Mol. Cell. Biol. 11:3307-3316, 1991), we used a variety of approaches to determine its actual subunit composition. Immunoprecipitation of epitope-tagged complex and reconstitution of the complex from in vitro-translated proteins demonstrated that only the Cyc8 and Tup1 proteins were present in the complex. Hydrodynamic properties showed that these proteins have unusually large Stokes radii, low sedimentation coefficients, and high frictional ratios, all characteristic of asymmetry which partly accounts for the apparent high molecular weight. Calculation of native molecular weights from these properties indicated that the Cyc8-Tup1 complex is composed of one Cyc8 subunit and four Tup1 subunits. This composition was confirmed by reconstitution of the complex from Cyc8 and Tup1 expressed in vitro and analysis by one-and two-dimensional gel electrophoresis.

Background: Mechanisms that redirect androgen receptor signaling to primarily support prostate tumor growth are poorly understood. Results: Prostate cancer cells were addicted to ELK1, which tethered AR to activate growth genes in hormone-dependent and castration-recurrent PC without ELK1 phosphorylation. Conclusion: ELK1 directs a critical arm of transcriptional growth signaling by AR that is preserved in CRPC. Significance: The ELK1-AR interaction offers a functionally tumor-selective drug target.