Biology subjects

Tschirpke, S.

Publications and source records attributed to Tschirpke, S..

4 recordsLinked to original sources

bioRxiv2024

Sortase A-mediated farnesylation of Cdc42 in vitro

Cdc42, a Rho-family GTPase, plays a pivotal role in establishing polarity in Saccharomyces cerevisiae by accumulating on the membrane at the site of bud emergence. Cdc42s ability to bind to membranes, mediated by prenylation, is essential for its function. Prenylation involves either the post-translational addition of a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group to Cdc42s C-terminus. One of the mayor challenges in studying the biophysical and biochemical interactions of Cdc42 at the polarity spot in vitro is obtaining prenylated Cdc42, due to labor-intensive and not easily reproducible traditional methods. Here, we present a streamlined, Sortase A-based approach to farnesylate Cdc42 in vitro. This method leverages E. coli-expressed Cdc42 with a Sortase A recognition motif, facilitating efficient and accessible farnesylation and purification using a purification tag-based strategy. The farnesylated Cdc42 retains functionality, as evidenced by GTP-dependent membrane binding, making it suitable for further biophysical and biochemical investigations. Additionally, our method can be easily adapted to yield geranyl-geranylated Cdc42.

biochemistry↗

bioRxiv2023

Quantification of GTPase cycling rates of GTPases and GTPase : effector mixtures using GTPase Glo™ assays

In different cellular activities like signal transduction, cell division, and intracellular transportation, small GTPases take on a vital role. Their functioning involves hydrolysing guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article we explain the application of a commercially accessible GTPase assay, known as the GTPase Glo assay by Promega, for the quantitative investigation of GTPase - effector interactions and the interplay between effectors. Basic ProtocolConducting GTPase assays with GTPase : effector protein mixtures using the GTPase Glo assay (Promega). Supporting Protocol 1Analysing GTPase assays to correlate the assay readout (luminescence) to amount of remaining GTP. Supporting Protocol 2Fitting GTPase assay data to obtain GTPase cycling rates.

biochemistry↗

bioRxiv2023

The GEF Cdc24 and GAP Rga2 synergistically regulate Cdc42 GTPase cycling

Cell polarity is a crucial biological process essential for cell division, directed growth, and motility. In Saccharomyces cerevisiae, polarity establishment centers around the small Rho-type GTPase Cdc42, which cycles between GTP-bound and GDP-bound states, regulated by GEFs like Cdc24 and GAPs such as Rga2. To dissect the dynamic regulation of Cdc42, we employed in vitro GTPase assays, revealing inverse concentration-dependent profiles for Cdc24 and Rga2: with increasing concentration, Cdc24s GEF activity is non-linear and oligomerization-dependent, which is possibly linked to the relief of its self-inhibition. In contrast, Rga2s GAP activity saturates, likely due to self-inhibition upon oligomerization. Together, Cdc24 and Rga2 exhibit a strong synergy driven by weak Cdc24 - Rga2 binding. We propose that the synergy stems from Cdc24 alleviating the self-inhibition of oligomeric Rga2. We believe this synergy contributes to efficient regulation of Cdc42s GTPase cycle over a wide range of cycling rates, enabling cells to resourcefully establish polarity. As Cdc42 is highly conserved among eukaryotes, we propose the GEF-GAP synergy to be a general regulatory property in other eukaryotes.

biochemistry↗

bioRxiv2023

A guide to the in vitro reconstitution of Cdc42 GTPase activity and its regulation

Cdc42 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 vitro reconstitutions 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 makes in vitro studies on Cdc42 less accessible to scientists that have a background different from biochemistry. We here present our experience with working with Cdc42 in vitro. We describe the recombinant expression and purification behaviour of 12 Cdc42, six Cdc42-mNeonGreenSW and four Cdc42-sfGFPSW constructs in E. coli. We explore Cdc42 dimerisation in vitro and 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 Cdc42 in vitro through describing Cdc42s properties in detail and examining assays that can be used to study its behaviour or act as activity checks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/538075v3_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18e6ecborg.highwire.dtl.DTLVardef@c068aorg.highwire.dtl.DTLVardef@1816ac5org.highwire.dtl.DTLVardef@188baa5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗