Mutational analysis of the Tup1 general repressor of yeast

Abstract

The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.