Post-translational regulation of Saccharomyces cerevisiae proteins tagged with the hormone-binding domains of mammalian
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ORIGINAL PAPER
H. Launhardt á T. Munder
Post-translational regulation of Saccharomyces cerevisiae proteins tagged with the hormone-binding domains of mammalian nuclear receptors Received: 17 January 2000 / Accepted: 7 June 2000 / Published online: 12 August 2000 Ó Springer-Verlag 2000
Abstract In the post-genome sequencing era the functional analysis of newly discovered proteins becomes more and more important. In this report we describe a genetic approach to the post-translational regulation of protein function in Saccharomyces cerevisiae by creating conditional lethal mutants. The yeast ORFs YDL139c, YDL147w, ERG3 and ERG11 were tagged with sequences encoding the hormone-binding domains of mammalian steroid receptors by PCR-mediated, targeted integration into the yeast genome. We found that the function of the chimeric proteins is regulated in a hormone-dependent way. This technique provides another important tool for the functional analysis of the yeast proteome. Key words Essential genes á Hormone-binding domain á Mammalian nuclear receptors á PCR-based integration á Saccharomyces cerevisiae
Introduction The major goal of the post-genome sequencing era is the assignment of functions to genes whose functions remain unknown. Many approaches are available for dissecting such functions, including the detection of protein/ protein-interactions (Hudson et al. 1997), subcellular localization of gene products (Burns et al. 1994), and genome-wide expression monitoring (Wodicka et al. 1997). The baker's yeast Saccharomyces cerevisiae provides a powerful model system in which to elucidate the
function of novel genes. About one-®fth of all yeast genes code for proteins which are essential for cell growth (Dujon 1996). Because of their importance to the cell knowledge of their activity is crucial to the understanding of the functional protein networks of the whole organism. However, by de®nition, no viable haploid knock-out mutants for essential yeast genes can be generated for further functional studies. The use of regulatable promoters allows tight repression and derepression of transcription (Mumberg et al. 1995; Belli et al. 1998), thus enabling determination of the terminal phenotypes of yeast mutants that lack particular essential genes. We have used a dierent strategy that permits posttranslational activation/inactivation of yeast proteins. This approach is based on tagging genes with sequences encoding the hormone-binding domains (HBDs) of mammalian steroid receptors. Due to the HBD moiety, the resulting chimeric protein is complexed with endogenous yeast heat-shock proteins in the absence of speci®c ligands, leading to the functional inactivation of the protein of interest. Ligands that are speci®c for the HBD cause the heat-shock factors to dissociate from the complex, thus giving rise to an active protein (Fig. 1). Using HBD-loxP-kanMx-loxP integration modules we have tagged several yeast genes of known and unknown function with the coding sequences for the HBDs of mammalian estrogen and glucocorticoid recept
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