Coordination of Cytokinesis and Cell Separation by Endosomal Targeting of a Cdc42-specific Guanine Nucleotide Exchange Factor inUstilago maydis

Abstract

The small GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in most eukaryotic cells. In Ustilago maydis, Cdc42 and the guanine nucleotide exchange factor (GEF) Don1 regulate cytokinesis and cell separation. Don1 belongs to the FGD1 family of Cdc42-specific GEFs that are characterized by a C-terminal lipid-binding FYVE domain. Although the FGD1/frabin family of Rho-GEFs is evolutionary conserved from fungi to mammals the role of the FYVE domain for its biological function is unknown. Here, we show that the FYVE domain is specific for phosphatidylinositol-3-phosphate (PtdIns(3)P) and targets Don1 to endosomal vesicles. During cytokinesis asymmetric accumulation of Don1-containing vesicles occurs at the site of septation. We could show that FYVE-dependent localization is critical for the function of Don1 at normal expression levels but can be compensated for by overexpression of Don1 lacking a functional FYVE domain. Our results demonstrate that endosomal compartmentalization of a Cdc42-specific exchange factor is involved in the coordination of cytokinesis and cell separation. INTRODUCTIONSmall GTP-binding proteins of the Rho/Rac/Cdc42 family act as molecular switches in various biological processes, such as cytoskeleton reorganization, vesicular trafficking, and cell polarity (Jaffe and Hall, 2005). They cycle between two states: the active GTP-bound state and the inactive GDP-bound state. The switching between these states is regulated by interaction with accessory proteins. GTPaseactivating proteins (GAPs) promote hydrolysis of GTP and thus trigger inactivation of the GTP-binding protein (Bernards and Settleman, 2004). Guanine nucleotide exchange factors (GEFs) catalyze the release of GDP by stabilizing the nucleotide-free transition state (Rossman et al., 2005). This results in exchange of GDP by GTP because the latter nucleotide is much more abundant in the cytoplasm. Most GEFs specific for members of the Rho/Rac/Cdc42 family are characterized by the presence of the catalytic dbl homology (DH) domain and an associated lipid-binding pleckstrin homology (PH) domain (Rossman et al., 2005). Although many different GEFs of the Dbl family are known, only little is known about their spatial and temporal regulation (Erickson and Cerione, 2004). Recent studies indicate that interaction of the PH domain with specific phosphoinositides seems to play a role not only for association to the plasma membrane but also for allosteric regulation of GEF activity (Baumeister et al., 2006). Most members of the Dbl family contain additional domains that contribute to spatial and temporal regulation of GEF signaling. The mammalian Rac1-specific GEF Tiam1 contains a second PH domain located in the N-terminal region (Habets et al., 1994). Members of the FGD1/Frabin family are characterized by a C-terminal FYVE domain (Pasteris et al., 1994;Obaishi et al., 1998). FYVE domains consist of a cysteine-rich Zinc finger region, which mediates endomembrane association by interaction with phosphatidylinositol phos...

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“…Most information comes from studies in model ascomycetes (41,46). To extend our understanding of fungal chitinases, we focused on the phytopathogenic basidiomycete U. maydis, whose complex life cycle comprises several morphological switches and represents a good model to study the .…”

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