Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor.
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.
Institut de Génétique et Microbiologie, CNRS URA D1354, Université Paris XI, Orsay, France.
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Garreau
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Jacquet
D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D002458:Cell Fractionation; D003600:Cytosol / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000502:physiology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D020690:GTPase-Activating Proteins; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins / Q000502:physiology*; D020666:ras-GRF1; D018909:src Homology Domains / Q000235:genetics
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C064960:IRA2 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D010749:Phosphoprotein Phosphatases; D019204:GTP-Binding Proteins; D018631:ras Proteins