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Related Experiment Videos

Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p.

J M Dolence1, L E Steward, E K Dolence

  • 1Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.

Biochemistry
|April 5, 2000
PubMed
Summary

The yeast RCE1 gene encodes a CaaX endoprotease crucial for Ras protein processing and localization. Rce1p is identified as a cysteine protease, making it a potential target for cancer therapeutics.

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Area of Science:

  • Molecular and Cellular Biology
  • Enzymology
  • Proteomics

Background:

  • Eukaryotic proteins with C-terminal CaaX motifs undergo prenylation, endoproteolysis, and carboxymethylation.
  • Two yeast genes, AFC1 and RCE1, encode CaaX endoproteases.
  • Rce1p is essential for the proteolytic processing of yeast Ras proteins.

Purpose of the Study:

  • To characterize the Saccharomyces cerevisiae RCE1 gene product (Rce1p) as a CaaX endoprotease.
  • To investigate the enzymatic properties and substrate specificity of Rce1p.
  • To evaluate Rce1p as a potential therapeutic target for cancer.

Main Methods:

  • Overexpression of Saccharomyces cerevisiae RCE1 under the GAL1 promoter.
  • Western blot analysis using specific antibodies to detect Rce1p.

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  • Enzyme inhibition assays with protease inhibitors, metal chelators, and heavy metals.
  • Site-directed mutagenesis of conserved amino acid residues in Rce1p.
  • Substrate selectivity assays using various prenylated CaaX peptides.

    • Main Results:

    • Rce1p is responsible for the endoproteolysis of yeast Ras proteins, and its absence impairs Ras2p plasma membrane localization.
    • Rce1p activity was significantly increased upon galactose-induced expression.
    • The enzyme is sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+).
    • Mutagenesis of conserved residues (H194A, E156A, C251A, H248A) resulted in inactive enzyme forms.
    • Rce1p demonstrated substrate selectivity for various prenylated CaaX peptides.

    Conclusions:

    • Rce1p is a critical cysteine protease involved in the post-translational modification of Ras proteins.
    • The mislocalization of Ras proteins due to impaired Rce1p function highlights its significance in cellular processes.
    • Rce1p represents a promising target for the development of novel cancer therapeutics.