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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.

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