The GEF Cdc24 and GAP Rga2 synergistically regulate Cdc42 GTPase cycling

Abstract Establishing cell polarity is vital for cells, as it is required for cell division, directed growth and secretion, and motility. A well-studied model organism for polarity establishment is Saccharomyces cerevisiae : here the small Rho-type GTPase Cdc42 exits the cytoplasm and accumulates in one spot on the cell membrane, marking the site of bud emergence. Due to redundancy and interconnection within the regulatory network surrounding Cdc42, the molecular mechanisms driving Cdc42 accumulation continue to be a subject of intense debate. In this study, we utilize a bulk in vitro GTPase assay to examine the GTPase cycle of Cdc42 in combination with two of its effectors - the GDP/GTP exchange factor (GEF) Cdc24 and GTPase activating protein (GAP) Rga2. We find that Cdc24’s GEF activity scales non-linearly with its concentration, which might be linked to Cdc24 di- or oligomerisation alleviating its autoinhibition. In contrast to Cdc24, Rga2 has an order of magnitude weaker GTPase cycle boosting effect which saturates at μM concentrations. Notably, Cdc24 combined with Rga2 leads to a large synergy in boosting Cdc42’s GTPase activity, which we hypothesise to be caused by the elevation of the Rga2 activity through Cdc24. Our data exemplifies a novel synergy within the regulatory network of Cdc42. This synergy contributes to efficient regulation of Cdc42’s GTPase cycle over a wide range of cycling rates, enabling cells to resourcefully establish polarity. As Cdc42 is highly conserved among eukaryotes, we suspect the GEF-GAP synergy to be a general regulatory property in other eukaryotes.