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Membrane‐anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor
Author(s) -
Garreau Herve´,
Geymonat Marco,
Renault Georges,
Jacquet Michel
Publication year - 1996
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1016/0248-4900(96)84771-x
Subject(s) - biology , saccharomyces cerevisiae , anchoring , microbiology and biotechnology , genetics , yeast , cognitive science , psychology
Summry— 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 ras1ras2 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.