Related Experiment Video
Updated: Jun 8, 2026
10:37
Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Analysis of Arf GTP-binding protein function in cells
Lee Ann Cohen1, Julie G Donaldson
1Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland, USA.
Current Protocols in Cell Biology
|September 21, 2010
Summary
This unit details methods for studying ADP-ribosylation factor (Arf) GTP-binding proteins and their regulators. It covers techniques to examine Arf protein localization, function, and activation status in cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- ADP-ribosylation factor (Arf) GTP-binding proteins are crucial regulators of intracellular transport and membrane dynamics.
- Arfs, along with Arf-like proteins (Arls), are conserved across eukaryotes and cycle between inactive GDP-bound and active GTP-bound states.
- Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) modulate Arf activity by controlling GTP binding and hydrolysis.
Purpose of the Study:
- To describe experimental techniques for investigating the functions of Arf and Arl proteins within cellular contexts.
- To provide protocols for assessing the cellular localization and functional roles of these GTPases.
- To present methods for measuring Arf activation and evaluating the functions of GEFs and GAPs.
Main Methods:
- Cellular localization studies using various imaging techniques.
- Functional assays to determine the roles of Arf and Arl proteins in cellular processes.
- Pull-down assays to measure the GTP-binding (activation) status of specific Arf proteins.
- Protocols for assessing the activity of GEFs and GAPs in modulating Arf function.
Main Results:
- Established methodologies for studying Arf and Arl protein localization and function in diverse cell types.
- A validated pull-down assay protocol for quantifying Arf activation in vivo.
- Approaches to investigate the regulatory roles of GEFs and GAPs on Arf GTPases.
Conclusions:
- The described techniques provide a comprehensive toolkit for dissecting the complex roles of Arf GTPases and their regulators in cellular signaling and trafficking.
- These methods facilitate the study of Arf-mediated processes in various biological systems, from yeast to humans.
- Understanding Arf function is critical for insights into fundamental cellular mechanisms and potential therapeutic targets.
More Related Videos
10:27Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
Published on: March 9, 2012
13:51Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
Published on: November 11, 2018
Related Concept Videos
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...
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...
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:
01:25Rab 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.
01:14Generation of Straight or Branched Actin Filaments
The straight or branched structure formation of actin filaments is controlled by nucleating proteins such as the formins and Arp2/3 complex. Formin-mediated assembly results in straight filaments, whereas Arp2/3 protein complex-mediated assembly results in branched actin filaments.
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
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...