The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes

• Published in: Molecular cell Vol. 31; no. 6; pp. 925 - 932
• Main Authors: Haruki, Hirohito, Nishikawa, Junichi, Laemmli, Ulrich K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-09-2008
• Subjects: Cell Nucleus - drug effects; Cell Nucleus - genetics; Cell Proliferation - drug effects; CELLBIO; DNA; Genes, Fungal; Genetic Complementation Test; Genetic Techniques; Mutagenesis - drug effects; Phenotype; Ribosomes - drug effects; Ribosomes - metabolism; RNA; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sirolimus - pharmacology; Time Factors; Transcription, Genetic - drug effects; CELLBIO; RNA; DNA
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2008.07.020

The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.