A guide to thein vitroreconstitution of Cdc42 GTPase activity and its regulation

Abstract

AbstractCdc42 is a small Rho-type GTPase and the main regulator of cell division in eukaryotes. It is surrounded by a large network of regulatory proteins. To understand the processes around cell division, in-depth understanding of Cdc42 and its regulation is required.In vitroreconstitutions are a suitable tool for such detailed mechanistic studies, as they allow a high level of control over the conditions and components used and. For these Cdc42 and its regulators need to be expressed, purified, and tested for their activity. There are many methods described for this, but their details, possible difficulties, and points of failure are rarely discussed. This makesin vitrostudies on Cdc42 less accessible to scientists that have a background different from biochemistry. We here present our experience with working with Cdc42in vitro. We describe the recombinant expression and purification behaviour of 12 Cdc42, six Cdc42-mNeonGreenSWand four Cdc42-sfGFPSWconstructs inE. coli. We explore Cdc42 dimerisationin vitroand assess its activity using GTPase Glo assays and Flag-pulldown assays. GTPase Glo assays turn out to be a reliable tool to quantitatively asses GTPase activities, wheareas pulldown experiments are more error prone. We find that most Cdc42 constructs, with the exception of those with an N-terminal Twin-Step-tag, show a similar GTPase activity and interaction with the GDP/GTP exchange factor Cdc24. We close with using enterokinase and TEV protease to generate untagged Cdc42. Enterokinase also cuts Cdc42 in an undesired position. TEV protease leads to the desired product, which retains its GTPase activity but shows a reduced Cdc24 interaction. The work presented here acts as a guide for scientists desiring to work with Cdc42in vitrothrough describing Cdc42’s properties in detail and examining assays that can be used to study its behaviour or act as activity checks.Graphical abstract