Anchor-Away: Efficient, Conditional Depletion of Nuclear Proteins in Saccharomyces cerevisiae.

For functional studies of proteins in cells, effective tools that trigger rapid and efficient inactivation of the proteins are indispensable. The anchor-away (AA) system is designed to deplete nuclear-localized proteins through cellular relocation. The target protein is sequestered by an anchor receptor protein from the nucleus to the cytoplasm. The depletion is achieved through the formation of a stable complex that is solely driven by the addition of rapamycin, between the FRB domain of human mTOR and the FKBP12-binding domain of the FRP protein. The target protein and the receptor protein are fused through genetic recombination with the FRB and FKBP12 domains, respectively, to mediate the interaction. The export of the nuclear protein of interest is executed by the receptor protein, i.e., ribosomal protein RPL13A, which is transiently imported into the nucleus and then exported to the cytoplasm within the pre-ribosome. The proteins of interest can be depleted within minutes although the depleting conditions need to be optimized. AA is a valuable tool to help dissect the biological functions of nuclear proteins, especially ones that are essential for cell viability. In this chapter, we describe the steps to construct the necessary strains and several biochemical and functional experiments to confirm the functional abrogation of target proteins by AA.