TY - JOUR
T1 - Auxin-inducible protein depletion system in fission yeast
AU - Kanke, Mai
AU - Nishimura, Kohei
AU - Kanemaki, Masato
AU - Kakimoto, Tatsuo
AU - Takahashi, Tatsuro S.
AU - Nakagawa, Takuro
AU - Masukata, Hisao
N1 - Funding Information:
We thank Drs. Haruhiko Takisawa for valuable discussion and Yoshinori Watanabe and Hiroshi Nojima for kindly providing derivatives of the adh1 promoter and S. pombe cDNA library, respectively. We also thank Mr. Tetsuya Handa, Ryota Ueda, Makoto Yoshida, Ooi LingFan and Ms. Shuqi Yan for construction of AID strains. This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Technology, Sports, and Culture, Japan, to H.M and by a Grant-in-Aid for JSPS Fellows to M.K.
PY - 2011/2/11
Y1 - 2011/2/11
N2 - 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), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.Conclusion: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.
AB - 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), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.Conclusion: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.
UR - http://www.scopus.com/inward/record.url?scp=79751532331&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79751532331&partnerID=8YFLogxK
U2 - 10.1186/1471-2121-12-8
DO - 10.1186/1471-2121-12-8
M3 - Article
C2 - 21314938
AN - SCOPUS:79751532331
SN - 1471-2121
VL - 12
JO - BMC Cell Biology
JF - BMC Cell Biology
M1 - 8
ER -