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First published online 16 November 2004
doi: 10.1242/jcs.01530

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Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that regulates cell wall ß-glucan biosynthesis through the GTPase Rho1p

Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, and Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain

View larger version (65K): [in a new window]   Fig. 1. Growth phenotypes of ehs2-1 mutant cells. (A) Morphology of ehs2-1 mutant cells grown at different temperatures. Differential interference-contrast micrographs of S. pombe wild-type (PN22) and ehs2-1 (GI 1) grown in YES liquid medium at 28°C or 37°C for 6 hours in the presence or absence of 1.2 M sorbitol (S). (B) Proportion of viable cells of the ehs2-1 mutant grown at different temperatures for the times indicated (with or without 1.2 M sorbitol) and plated on rich medium at 28°C.

View larger version (37K): [in a new window]   Fig. 2. Complementation of the ehs2-1 thermosensitive and hypersensitive phenotypes by plasmids pAL-rgf3 (pYS10) and pAL-rgf1 (pYS8). GI1 (h+ leu1-32, ehs2-1) cells were transformed with pAL-rgf3, pAL-rgf1 or pAL (empty plasmid). (A) Transformants were selected in MM and the temperature-sensitive phenotype was scored by incubating the cultures for 4 hours at 37°C. Differential-interference-contrast images are shown. (B) Transformants were streaked out on YES plates in the presence or absence of echinocandin (Ech) (1 µg ml-1) or Calcofluor White (Cfw) (1 mg ml-1). Plates were incubated at 28°C for 4 days. (C) Schematic illustration of structural features analysed by the SMART program (Letunic, 2002) (http://smart.embl-heidelberg.de/). Domains are indicated: CNH, citron homology domain (this acts as a regulatory domain and could be involved in macromolecular interactions); DEP, domain of unknown function present in signalling proteins that contain PH, RasGEF, RhoGEF, RhoGAP, RGS or PDZ domains; PH, pleckstrin-homology domain; RhoGEF, domain conserved among GEFs for Rho/Rac/Cdc42-like GTPases. (D) Alignment of predicted amino acid sequence of ehs2-1 with the corresponding region of known GEF proteins from different organisms (S. pombe Scd1, Caenorhabditis elegans unc-73, human Dbl, human Abr, human Bcr, mouse Vav and S. cerevisiae Cdc24). Multiple sequence alignments were performed using the ClustalW program. The site of mutation is located within the RhoGEF domain in a highly conserved region called CR3 and is marked with `611' over the predicted amino-acid sequence of Ehs2-1p. Asterisks indicate identical amino acids among all identified gene products. (.) and (:) indicate well-conserved and highly conserved amino acids, respectively.

View larger version (68K): [in a new window]   Fig. 3. Rgf3p is essential for cell viability and depletion of Rgf3p leads to a lysis phenotype similar to the depletion of Rho1p. (A) Genomic organization of the rgf3+ and rgf1+ loci, and deletion strategy for rgf3+ disruption. The direction of transcription is indicated by an arrow. Tetrads from a rgf3::ura4+/rgf3+ strain dissected on YES medium and incubated at 28°C for 4 days. (B) Terminal phenotype of rgf3-null mutants. Spores prepared from the rgf3::ura4+ strain were inoculated in MM lacking uracil and germinated for 18 hours. Cells were stained with rhodamine-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively (top left) and with Calcofluor White (Cfw) to visualize the cell-wall material (top right). Spores with the wee1-50 rgf3 and sid2-250 rgf3 double mutations (prepared from strains YSM654 and YSM656, respectively) were inoculated in YES medium and germinated for 14 hours at 25°C and then for 6 hours at 36°C. Cells were stained with Hoechst and Cfw (bottom). (C) Lethal phenotype of the P81 nmt-rgf3 and P41 nmtrho1 shut-off mutants. Cells grown at 28°C in MM were supplemented with thiamine to repress the nmt promoter. Nomarsky micrographs were taken after 12 hours in MM with or without thiamine. (D) Growth phenotypes of P81 nmt-rgf3 and P41 nmt-rho1mutants under different growth conditions. Strains VT88 (81 nmt-rgf3+ + pREP81X) and PPG217 (rho1 + pREP41X nmt-rho1+) were streaked onto several plate media (YES, YES + Sorbitol and MM-leu) and the plates were incubated for 3 days at 28°C. The nmt promoter is off in rich medium (YES) and on in MM. Strain VT88 carried pREP81X, an empty plasmid, to allow cells to grow in MM-leu.

View larger version (86K): [in a new window]   Fig. 4. Suppression of the echinocandin-hypersensensitive growth phenotype of the ehs2-1 mutant by overexpression of rho1+. MS38 (rgf3+) was transformed with pREP3X (empty vector) and GI 1 (ehs2-1/rgf3) was transformed with pREP3X-rho1 (rho1+), pREP3X-rho2 (rho2+), pREP3X-rho3 (rho3+), pREP3X-rho4 (rho4+), pREP3X-rho5 (rho5+), pREP3X-cdc42 (cdc42+) and pREP3X (empty vector). Transformants were streaked onto MM, MM plus thiamine, MM plus 1.5 µg ml-1 echinocandin and MM plus thiamine and 1.5 µg ml-1 echinocandin plates, and incubated at 32°C for 4 days.

View larger version (60K): [in a new window]   Fig. 5. Rgf3p is a specific Rho1-GEF. (A) Rgf3 binds directly to the GDP-bound form of Rho1p (Rho1-T20N). Y190 cells expressing the indicated proteins were cultured on a SD plate with histidine (left) or without histidine plus 40 mM 3AT (right) at 30°C for 3 days. (B) The Rgf3p level modulates the amount of GTP-bound Rho1p in vivo. Wild-type (MS38) cells expressing pREP4X or pREP4X-rgf3, and ehs2-1 (GI 1) mutant cells were transformed with either pREP3XHA-rho1 or pREP3X-HA-rho2. GTP-Rho1p or GTP-Rho2p were pulled down from the cell extracts with GST-C21RBD and blotted against 12CA5, anti-HA monoclonal antibody.

View larger version (57K): [in a new window]   Fig. 6. Phenotypes of Rgf3p overexpression. (A) Micrographs of Calcofluor White (Cfw) stained wild-type cells transformed with pREP3X (empty plasmid) or pREP3X-rgf3+ (rgf3+ overexpression) grown without thiamine for 20 hours. (B) In vitro glucan synthase (GS) activity assayed with the membrane fraction of wild-type cells (MS38) transformed with pREP3X, pREP4X-rgf3 (rgf3+ overexpression), pREP3X-rho1 (rho1+ overexpression) or both pREP4X-rgf3 and pREP3X-rho1 (rgf3+ and rho1+ overexpression). Extracts were prepared from cells grown in MM without thiamine at 32°C for 18 hours. Specific activity is expressed as milliunits per mg protein. Values are the means of at least three independent experiments with duplicated samples, and error bars represent standard deviations (s.d.). (C) Cell-wall composition in cells that overexpress rgf3+. The relative levels of [14C]-glucose radioactivity incorporated into each cell-wall polysaccharide are shown for the same strains as above: wild-type (MS38) transformed with pREP3X, pREP4X-rgf3 (rgf3+ overexpression), pREP3X-rho1 (rho1+ overexpression) or both at the same time. Cells were grown in the absence of thiamine for 18 hours and then [14C]-glucose was added 6 hours before harvesting the cells. Values are the means of three independent experiments with duplicate samples. Standard deviations for total carbohydrate values are shown.

View larger version (66K): [in a new window]   Fig. 7. Rgf3p localizes to the septum. (A) Wild-type cells were transformed with an integrative plasmid expressing EGFP-rgf3+ under its own promoter (strain VT128). Cells were grown at 28°C. (Top) EGFP-Rgf3p localization in a population of living cells at different stages of the cell cycle is shown in the centre. The corresponding differential interference contrast images are shown on the left and Calcofluor White (Cfw) staining is shown on the right. (Bottom) A square with cells that had not yet developed a visible septum is enlarged. These cells already showed EGFP-Rgf3p dots of fluorescence. (B) The rgf3 mRNA levels were followed by northern-blot analyses in a cdc25-22 block-release experiment. RNA samples were collected every 20 minutes along two consecutive cell cycles after release to 25°C. The blot was probed for rgf3 and for act1, the latter as an mRNA loading control.

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