Related Experiment Video
Updated: May 31, 2026
13:51
Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
Published on: November 11, 2018
Dimeric plant RhoGAPs are regulated by its CRIB effector motif to stimulate a sequential GTP hydrolysis
Antje Schaefer1, Mandy Miertzschke, Antje Berken
1Department of Structural Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn Str. 11, 44227 Dortmund, Germany.
Journal of Molecular Biology
|July 5, 2011
Summary
Plant RopGAPs, GTPase-activating proteins for ROPs, utilize a unique CRIB motif for efficient activity. Dimeric RopGAP2 exhibits sequential GTP hydrolysis, a mechanism likely caused by steric hindrance from bound ROP molecules.
Area of Science:
- Plant molecular biology
- Cell signaling
- Protein-protein interactions
Background:
- RopGAPs are plant-specific GTPase-activating proteins (GAPs) for Rho proteins (ROPs).
- The largest RopGAP family uniquely combines a RhoGAP domain with a Cdc42/Rac interactive binding (CRIB) motif.
- The molecular mechanism and regulatory role of the CRIB motif in RopGAP activity are largely unknown.
Purpose of the Study:
- To analyze the kinetics of RopGAP-mediated GTP hydrolysis of ROPs.
- To investigate the role of the CRIB motif and catalytic residues in RopGAP activity.
- To elucidate the mechanism behind the sequential GTP hydrolysis observed in dimeric RopGAP2.
Main Methods:
- Kinetic analysis of GTP hydrolysis using wild-type and mutant RopGAP2 from Arabidopsis thaliana.
- Site-directed mutagenesis of the catalytic Arg159 residue and the CRIB motif.
- Characterization of RopGAP2-ROP complex kinetics.
Main Results:
- Efficient RopGAP2 activity requires both the catalytic Arg159 and the CRIB motif for high-affinity, specific ROP binding.
- Dimeric RopGAP2 exhibits unique sequential stimulation of ROP·GTP hydrolysis with a 10-fold rate difference between active sites.
- CRIB point and deletion mutants suggest steric hindrance from Arg fingers and/or CRIB motifs induces the sequential mechanism.
Conclusions:
- The CRIB motif is essential for RopGAP2's catalytic efficiency and specificity.
- Dimeric RopGAP2 employs a sequential hydrolysis mechanism, likely regulated by steric factors.
- This study provides novel insights into the molecular mechanism of plant RopGAP regulation.
Related Concept Videos
01:24Small GTPases - Ras and Rho
Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Three regulatory proteins control their activity:
01:22Activation and Inactivation of G Proteins
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high affinity and are together...
01:25Rab Cascades
Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
01:14Rab Proteins
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
01:25Cell Signaling in Plants
Plant cells communicate to coordinate their cycle of growth, flowering and fruiting, and activities in roots, shoots, and leaves in response to the changing environmental conditions. Plant signaling is distinct from animal signaling. Plants primarily utilize enzyme-linked receptors, whereas the largest class of cell-surface receptors in animals are G-protein coupled receptors (GPCRs). Unlike animals, receptor tyrosine kinases are rare in plants. Instead, plants have a diverse class of...
02:14GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...