Temperature-sensitive Control of Protein Activity by Conditionally Splicing Inteins

Nat Biotechnol. 2004 Jul;22(7):871-6. doi: 10.1038/nbt979. Epub 2004 Jun 6.

Abstract

Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism
  • Inteins / genetics*
  • Membrane Proteins / analysis
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Protein Splicing / genetics*
  • Receptors, Notch
  • Repressor Proteins / analysis
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / analysis
  • Saccharomyces cerevisiae Proteins / biosynthesis
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Temperature*
  • Transcription Factors / biosynthesis
  • Transcription Factors / genetics*
  • Vacuolar Proton-Translocating ATPases / genetics

Substances

  • DNA-Binding Proteins
  • Drosophila Proteins
  • GAL4 protein, S cerevisiae
  • GAL80 protein, S cerevisiae
  • Membrane Proteins
  • N protein, Drosophila
  • Receptors, Notch
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Vacuolar Proton-Translocating ATPases