Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor

Biol Cell. 1996;86(2-3):93-102. doi: 10.1016/0248-4900(96)84771-x.

Abstract

The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180-kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3' part of the gene (codons 877-1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459-1471) and no consensus sequence for post-translational acylation. The SH3 domain present in the N-terminal part of Cdc25p is not involved nor required for membrane localization, since the N-terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C-terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1 ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441-1552.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / genetics
  • Cell Fractionation
  • Cytosol / chemistry
  • Fluorescent Antibody Technique
  • Fungal Proteins / physiology
  • GTP-Binding Proteins / chemistry*
  • GTP-Binding Proteins / genetics
  • GTPase-Activating Proteins*
  • Gene Deletion
  • Gene Expression Regulation / physiology
  • Immunoblotting
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Mutagenesis / physiology
  • Phosphoprotein Phosphatases / chemistry*
  • Phosphoprotein Phosphatases / genetics
  • Plasmids / genetics
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins*
  • ras Proteins / physiology*
  • ras-GRF1
  • src Homology Domains / genetics

Substances

  • Cell Cycle Proteins
  • Fungal Proteins
  • GTPase-Activating Proteins
  • IRA2 protein, S cerevisiae
  • Membrane Proteins
  • Saccharomyces cerevisiae Proteins
  • ras-GRF1
  • Phosphoprotein Phosphatases
  • GTP-Binding Proteins
  • ras Proteins