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 TUP1 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 β subunit of heterotrimeric 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-TUP1 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 Δcyc8 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 TUP1 genes.