Email updates

Keep up to date with the latest news and content from BMC Cell Biology and BioMed Central.

Open Access Highly Accessed Methodology article

Auxin-inducible protein depletion system in fission yeast

Mai Kanke1, Kohei Nishimura12, Masato Kanemaki12, Tatsuo Kakimoto1, Tatsuro S Takahashi1, Takuro Nakagawa1 and Hisao Masukata1*

Author affiliations

1 Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan

2 Current Address: National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan

For all author emails, please log on.

Citation and License

BMC Cell Biology 2011, 12:8  doi:10.1186/1471-2121-12-8

Published: 11 February 2011

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 (

    i
mproved-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.