Auxin-inducible protein depletion system in fission yeast

BMC Cell Biol. 2011 Feb 11;12:8. doi: 10.1186/1471-2121-12-8.

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), 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle / drug effects*
  • Cell Cycle / genetics
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Chromatin / genetics
  • Chromatin / metabolism
  • DNA Replication / drug effects
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • F-Box Proteins / genetics
  • F-Box Proteins / metabolism
  • Gene Expression / drug effects*
  • Gene Expression / genetics
  • Indoleacetic Acids / pharmacology
  • Minichromosome Maintenance Complex Component 4
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Pol1 Transcription Initiation Complex Proteins / genetics
  • Pol1 Transcription Initiation Complex Proteins / metabolism
  • Polyubiquitin / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • SKP Cullin F-Box Protein Ligases / genetics
  • SKP Cullin F-Box Protein Ligases / metabolism
  • Schizosaccharomyces / cytology
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Transcriptional Activation
  • Transformation, Genetic

Substances

  • CDC45 protein, S pombe
  • Cell Cycle Proteins
  • Chromatin
  • DNA-Binding Proteins
  • F-Box Proteins
  • Indoleacetic Acids
  • Nuclear Proteins
  • Plant Proteins
  • Pol1 Transcription Initiation Complex Proteins
  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins
  • Polyubiquitin
  • SKP Cullin F-Box Protein Ligases
  • SKP1 protein, S pombe
  • Minichromosome Maintenance Complex Component 4
  • mcm4 protein, S pombe