Characterization of Rho GTPase GAP/GEF modules in the ascomycete Neurospora crassa

Rho (Ras homologue) GTPases are members of the Ras superfamily and known as key players in highly conserved signaling pathways regulating cellular processes like metabolism, survival, differentiation, vesicle transport and morphogenesis. The cycling between active and inactive states is essential for full signaling activity of the Rho GTPase (Barale et al., 2006, Vanni et al., 2005). Regulation of Rho GTPases is achieved by GTPase activating proteins (GAPs) leading to an inactive Rho GTPase and guanine nucleotide exchange factors (GEFs) that activate the small Rho GTPase (Jaffe & Hall, 2005b). Their molecular function during polar tip growth in the model mold Neurospora crassa is poorly understood. The filamentous fungi N. crassa encodes six Rho GTPases named RHO-1 to RHO-4, CDC-42 and RAC. Previous phylogenetic and domain structure analyses identified ten potential GAPs in N. crassa. Since LRG-1 has already been described as Rho1 specific GAP (Vogt & Seiler, 2008), the nine remaining GAPs were further analyzed. In vitro GAP activity assays determined target specificity of four GAPs (RGA-1 to RGA-4) towards specific Rho GTPases. In vitro GAP activity assays depicted dual specificity of RGA-1 to RHO-1 and RHO-4. While in vitro pull-down assays confirmed the RGA-1 interaction with RHO-4, further in vivo RGA-1-GFP microscopy studies revealed a localization pattern at the forming septa, comparable to the localization of GFP-RHO-1. Together with the Δrga-1 defect in septation (irregular clusters and curved septa), which defects phenocopy those of the dominant-active Rho4, these data imply a role of RGA-1 during septation as dual specific GAP of RHO-4 and RHO-1 in N. crassa. In this study, a comparative phenotypic characterization of nine GAPs was elaborated, which show predominantly marginal phenotypes. Furthermore, any of the GAPs revealed in stress tests hypersensitivity to Congo Red, Calcoflour White (interfering cell wall assembly) and latrunculin A (actin polymerization inhibitor) a preliminary hint to their potential function in the regulation of the actin cytoskeleton and/or proper function of the cell wall. RGA-2, RGA-3 and RGA-4 were assigned to be involved in CDC-42, RAC and RHO-3 regulation. To investigate functional relations three double deletion mutants were generated. The double deletion of Δrga-2;Δrga-3 leads to a phenotype resembling the Δrga-2 mutant, whereas the Δrga-3;Δrga-4 double deletion phenocopied the Δrga-4 deletion mutant and in both strains no additional morphological characteristics were identified. However, the Δrga-2;Δrga-4 double deletion was characterized by more severe morphological defects than the single deletion mutants. These results suggest overlapping or partially redundant functions for RGA-2 and RGA-4 in regulation of Rho GTPases in relation to maintain polar growth in N. crassa. Dock180 and Elmo1 were first described in mammals as bipartite GEF of Rac1 (Cote & Vuori, 2002, Jaffe & Hall, 2005b). So far, in N. crassa only CDC-24 was described to function as dual GEF of the RAC-1-CDC-42 module (Araujo-Palomares et al., 2011). Interaction studies verified that ELMO and DOCK form a complex and both proteins interact with RAC, but not with CDC-42 in N. crassa. Phenotypical characterization of Δdock revealed a bulgy hyperbranched phenotype, whereas the Δelmo phenotype was reminiscent to the Δdock phenotype, but not identical. The defects of the Δdock mutant were phenocopied by the Δdock;Δelmo double deletion. In vivo microscopy studies identified subapical GFP-DOCK localization in a patchy membrane associated manner. The CRIB-GFP reporter construct revealed additional subapical membrane associated localization in Δdock. These results indicate a role of DOCK in polar growth in N. crassa. Taken together, this analysis in N. crassa will establish a more comprehensive understanding how important the spatio-temporal regulation of Rho GTPases is.