The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex.

Abstract

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...

A Complex Composed of Tup1 and Ssn6 Represses Transcription in Vitro

“…The peak fractions were pooled and further fractionated by gel filtration. As determined by Western immunoblotting, Ssn6HIS and Tup1 were among the first proteins to flow through the filtration column, consistent with the results of Williams et al (18) that these proteins are present together in a large complex (Fig. 1A, fraction 9 and 10).…”

“…Fig. 1C shows the purified proteins on a silver- Determination of the Size of the Ssn6-Tup1 Complex-It has been suggested that the previous estimate of the molecular mass of the Ssn6-Tup1 complex of 1.2 MDa may be an overestimate if the complex is elongated or extended (18). Glycerol gradient sedimentation in conjunction with gel filtration can be used to estimate the molecular mass of a molecule with greater accuracy than can either method alone (32).…”

“…Tup1 and Ssn6 interact directly in vitro and are found associated in a large complex in yeast extracts estimated at 1.2 MDa (18). The size of this complex suggests that it consists of many protein subunits.…”

LncRNAs: Bridging environmental sensing and gene expression

“…In the presence of glucose, the GAL cluster is repressed via association of glucose-dependent transcription factors, Mig1 and Nrg1, and the associated Tup1/Cyc8 (Tup1/Ssn6) co-repressor complex. 42,43 In the presence of galactose, the Gal4 transcriptional activator associates with GAL genes and facilitates transcriptional activation by promoting recruitment of co-activators and RNA polymerase II (RNA P II). 44 The third transcriptional state, called the "derepressed" or "non-induced" state, occurs when yeast are grown in the presence of raffinose.…”

Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

“…WD repeats typically contain a GH dipeptide 11-24 residues from the N-terminus, and the WD dipeptide at the C-terminus. Proteins with WD repeats have diverse functions, such as signal transduction, RNA processing, transcription, cytoskeleton assembly and mitotic-spindle formation, regulation of vesicle formation and vesicular trafficking, control of various aspects of cell division, and the regulation of sulfur metabolism in fungi [9][10][11][12][13][14][15][16][17][18].…”