AgRho1a. Furthermore, it enables the mutated allele to complement the lethality of an AgRHO1b deletion. In summary, our findings show that a simple mutation in the switch I region of a GTP-binding protein can change its affinity towards its GAPs, which finally leads to a decoupling of very similar protein function without impairing effector interaction.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/7/1065/DC1 Journal of Cell Science 1066 organization of the AgRHO1 genes together with the neighboring genes in A. gossypii and S. cerevisiae is shown in Fig. 1A. The two copies very probably originate from a tandem-duplication event.Little is known about the function of ABR182W. Deletion mutants have been reported to display a weak lysis phenotype and a weak sensitivity to the chitin-binding dye Calcofluor White (Walther and Wendland, 2005). Here, we studied the differences in function of a pair of duplicated RHO-type GTP-binding proteins in A. gossypii and compared them with their single homolog in budding yeast. Surprisingly, we found that a single residue in AgRho1a is altered in the highly conserved switch I region. This residue contributes to divergence of protein function and alters GAP specificity without impairing effector interaction. ResultsThe lytic phenotype of Agrho1a⌬ is temperature-dependent We first characterized Rho1 protein function in A. gossypii to test whether protein function diverged. An alignment of the protein sequences shows that AgRho1a is 73.9% identical to AgRho1b and 75.6% identical to ScRho1, whereas the latter two proteins share the highest number of identical residues (83.3%) (Fig. 1B). As a first step towards defining the functional difference between AgRHO1a and AgRHO1b, we compared the phenotypes of deletion mutants. Although Agrho1aΔ formed a mature mycelium that grew slightly slower than the wild-type control, deletion of AgRHO1b resulted in cells that never reached the state of a mature mycelium and died because of cell lysis (Fig. 1C). Agrho1aΔ cells also lysed but only few hyphae were affected. Fig. 1D shows an example of lysing hyphae taken from Movie 1 (supplementary material). We also monitored the strain deleted for AgRHO1a at different growth temperatures. Lysis of adult mycelia was quantified by an assay that visualizes the release of cytoplasmic alkaline phosphatase upon lysis by a color reaction (Fig. 1E, for details, refer to Materials and Methods). An Agslt2⌬ strain was used as a lysis control. AgSLT2 is the homolog of the S. cerevisiae MAP kinase responsible for maintenance of cellular integrity (Torres et al., 1991). In contrast to Agrho1b deletion mutants, Agslt2 deletions can form a mature mycelium despite extensive lysis. Hyphae at the colony border grow normally and cannot be distinguished microscopically from wild type but the inner, older parts of the mycelium show extensive lysis. At 25°C and 30°C, Agrho1a deletion mutants released about half as much alkaline phosphatase as the Agslt2 deletion mutants. Surprisingly,...