auxin-inducible protein depletion system in fission yeast在裂殖酵母auxin-inducible蛋白质消耗系统.pdfVIP

Kanke et al. BMC Cell Biology 2011, 12:8 /1471-2121/12/8 METHODOLOGY ARTICLE Open Access Auxin-inducible protein depletion system in fission yeast 1 1,2 1,2 1 1 1 Mai Kanke , Kohei Nishimura , Masato Kanemaki , Tatsuo Kakimoto , Tatsuro S Takahashi , Takuro Nakagawa , Hisao Masukata1* Abstract Background: Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe. Results: We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly- ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system),