Development of AlissAID system targeting GFP or mCherry fusion protein.

Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells. Author summary: Budding yeast has been widely studied as a genetic model organism for eukaryotic species. Targeted degradation systems, in which conditional-control of target protein degradation, are used to examine protein function. The auxin-inducible degron (AID) system is one such system that rapidly disrupts target proteins in an auxin (IAA)-dependent manner. In this system, high concentrations of auxin and AID-tagging to the target proteins are necessary for degradation. Here, we developed the Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system, which enables to degrade target proteins with nM concentration of a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In this system, we can use popular fluorescent proteins GFP or mCherry as degradation tags and avoid AID-tagging to the target endogenous gene. As GFP Clone Collection is commercially available in budding yeast, it is easy to generate conditional mutants in AlissAID system. AlissAID system would be a powerful tool of genetic analysis in eukaryotic species including budding yeast. [ABSTRACT FROM AUTHOR]