Efficient protein depletion by genetically controlled deprotection of a dormant N-degron

Mol Syst Biol. 2009;5:267. doi: 10.1038/msb.2009.25. Epub 2009 Apr 28.

Abstract

Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endopeptidases / genetics*
  • Phenotype
  • Protein Processing, Post-Translational*
  • Protein Stability
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Saccharomyces cerevisiae Proteins
  • Endopeptidases
  • TEV protease