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Updated: Jun 17, 2026
09:58
RhoC GTPase Activation Assay
Published on: August 22, 2010
Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs
Eiko Kanno1, Koutaro Ishibashi, Hotaka Kobayashi
1Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Traffic (Copenhagen, Denmark)
|January 15, 2010
Summary
Researchers identified novel Rab-binding proteins, including three GTPase-activating proteins (GAPs), that regulate membrane trafficking. One protein, centaurin beta2, interacts with Rab35 via its ANKR domain, influencing neurite outgrowth.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The Rab family, small GTPases, are crucial for membrane trafficking.
- Most mammalian Rab effectors remain unidentified, hindering understanding of their functions.
Purpose of the Study:
- To systematically identify novel Rab effectors in mammalian cells.
- To characterize the interaction and function of newly identified Rab-binding proteins.
Main Methods:
- Glutathione S-transferase (GST) pull-down assays using 60 mammalian Rabs.
- Mass spectroscopic analysis of Rab-binding proteins from five cell/tissue lysates.
Main Results:
- Identified 21 Rab-binding proteins, including three GTPase-activating proteins (GAPs): mKIAA1055/TBC1D2B, GAPCenA/TBC1D11, and centaurin beta2/ACAP2.
- Discovered that centaurin beta2 binds Rab35 via its ankyrin repeat (ANKR) domain, not the GAP domain.
- Demonstrated that centaurin beta2's ANKR domain is essential for plasma membrane localization and Rab35-dependent neurite outgrowth via Arf6 inactivation.
Conclusions:
- Novel Rab-binding proteins, including GAPs, were identified, expanding the known Rab effector network.
- A non-GAP domain interaction between centaurin beta2 and Rab35 suggests a new mode of Rab-GAP regulation.
- Centaurin beta2 plays a role in neurite outgrowth, highlighting the functional significance of Rab-effector interactions.
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