Rho GTPases are small proteins present in all eukaryotic cells, from yeast to mammals, with a function in actin organization and morphogenetic processes. Schizosaccharomyces pombe Rho4 is not essential but it displays a role during cell separation at high temperature. In fact, Rho4 is involved in the secretion of the hydrolytic enzymes that are required for cell septum degradation during this process. In rho4D cells, vesicles accumulate in the septum area and the glucanases Eng1 and Agn1 are not secreted to the culture medium. The localization of Eng1 and Agn1 depends on the exocyst and the septins. The exocyst is a conserved multiprotein complex important for the targeting and fusion of Golgi-derived vesicles with the plasma membrane. Septins are a family of GTP-binding proteins conserved in eukaryotes that function during cytokinesis. Here we show that Rho4 is required for the proper localization of the exocyst and septins at high temperature. Moreover, pull-down experiments demonstrate that Rho4 can interact with exocyst subunits, such as Sec8 and Exo70, and septin proteins, such as Spn3. We observe that Sec8 preferentially binds to activated GTP-Rho4, suggesting that Sec8 could be an effector of this GTPase. We propose that the interaction of Rho4 with the exocyst and septins confers a precise regulation for the secretion of glucanases at the appropriate place and time during the cell cycle.

Rho GTPases control polarized cell growth and are well-known regulators of exocytic and endocytic processes. Cdc42 is an essential GTPase, conserved from yeast to humans, that is critical for cell polarization. Cdc42 is negatively regulated by the GTPase-activating proteins (GAPs) and the GDP dissociation inhibitors (GDIs), and positively regulated by guanine nucleotide exchange factors (GEFs). Cdc42 GTPase can be found in a GTP- or GDP-bound state, which determines the ability to bind downstream effector proteins and activate signalling pathways. Only GTP-bound Cdc42 is active. In this study we have analysed the localization of the different nucleotide-bound states of Cdc42 in Schizosaccharomyces pombe: the wild-type Cdc42 protein that cycles between an active and inactive form, the Cdc42G12V form that is permanently bound to GTP and the Cdc42T17N form that is constitutively inactive. Our results indicate that Cdc42 localizes to several membrane compartments in the cell and this localization is mediated by its C-terminal prenylation. Constitutively active Cdc42 localizes mainly to the plasma membrane and concentrates at the growing tips where it is considerably less dynamic than wild-type or GDP-bound Cdc42. Additionally we show that the activation state of Cdc42 also participates in the regulation of its protein levels mediated by endocytosis and by the exocyst complex.