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.

Quantification of GTPase Cycling Rates of GTPases and GTPase:Effector Mixtures Using GTPase Glo Assays

“…9 of Supporting Information S8). Assay variations are typically very small (reflected as c corr ≈ 1.0) (Tschirpke, Daalman et al., 2023; Tschirpke, van Opstal et al., 2023). We recommend excluding assays that show vastly different protein activities (reflected in a c corr much larger or much smaller than 1), as these indicate that the GTPase behavior/assay conditions are unusual.…”

“…Our recommended procedure is explained below using as an example a GTPase assay series investigating the cycling of a GTPase and the effects of both a GEF and a GAP, alone and in combination (example 2; Fig. 2; Tschirpke, Daalman et al., 2023).…”

“…If executed properly, the assay leads to reliable and reproducible results (e.g., Tschirpke, Daalman et al., 2023; Tschirpke, van Opstal et al., 2023).…”

“…The fit is a phenomenological description of the data and does not reveal the origin of the nonlinearity. One possibility could be dimerization, with Cdc24 dimers exhibiting increased activity (Tschirpke, Daalman et al., 2023). Values of k 3,Cdc24 are shown in Data_summary.xlsx as Parameter 2 for assays in which Cdc24 is varied (Varied Protein 1).…”

“…We do not advise using this protocol if single GTPase steps are to be examined in‐depth, but rather when the effects of a protein on the entire GTPase cycle are of interest. This method is illustrated using a basic GTPase assay with human Ras (GTPase) (example 1) and an elaborate GTPase assay using Saccharomyces cerevisae Cdc42 (GTPase), Cdc24 (GEF), and Rga2 (GAP) (example 2) (Tschirpke, Daalman et al., 2023). Table 1 summarizes how these examples are used to illustrate the protocols described.…”