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Related Experiment Videos

Heat-inducible degron: a method for constructing temperature-sensitive mutants

R J Dohmen1, P Wu, A Varshavsky

  • 1Division of Biology, California Institute of Technology, Pasadena 91125.

Science (New York, N.Y.)
|March 4, 1994
PubMed
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Researchers created a temperature-sensitive (ts) dihydrofolate reductase (Arg-DHFRts) that acts as a heat-inducible degradation signal. This novel tool allows for the creation of ts mutants without traditional mutation screening.

Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Protein Degradation

Background:

  • The N-end rule pathway dictates protein stability based on the N-terminal amino acid.
  • Arginine is typically a destabilizing residue under the N-end rule.
  • Dihydrofolate reductase (DHFR) is essential for nucleotide synthesis.

Purpose of the Study:

  • To develop a novel method for generating temperature-sensitive (ts) mutants.
  • To investigate the utility of a modified dihydrofolate reductase (Arg-DHFR) as a conditional degradation signal.
  • To explore the application of Arg-DHFRts in creating ts phenotypes for essential yeast genes.

Main Methods:

  • Engineering a temperature-sensitive (ts) derivative of arginine-bearing dihydrofolate reductase (Arg-DHFR).

Related Experiment Videos

  • Assessing the stability of Arg-DHFRts at permissive (23°C) and nonpermissive (37°C) temperatures.
  • Creating fusion proteins of Arg-DHFRts with essential yeast proteins (Ura3 and Cdc28).
  • Evaluating the resulting ts phenotypes conferred by the Arg-DHFRts fusions.

    • Main Results:

    • A ts derivative of Arg-DHFR (Arg-DHFRts) was identified, stable at 23°C but rapidly degraded at 37°C via the N-end rule pathway.
    • Fusion of Arg-DHFRts to Ura3 or Cdc28 resulted in specific temperature-sensitive phenotypes for these proteins.
    • This demonstrates that Arg-DHFRts functions as a heat-inducible degradation signal.

    Conclusions:

    • Arg-DHFRts serves as an effective heat-inducible degradation signal.
    • This system provides a new strategy for generating temperature-sensitive mutants in Saccharomyces cerevisiae.
    • The approach bypasses the need for traditional screening methods to identify ts mutations.