ARL3 activation requires the co-GEF BART and effector-mediated turnover

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• ADP-ribosylation factor-like 3 (ARL3) is a G-protein essential for ciliary trafficking of lipid-modified proteins.
• ARL3's activation mechanism by its guanine exchange factor (GEF), ARL13B, is not fully understood.
• BART, an ARL3 effector, is linked to ciliopathies, but its specific role in ARL3 interaction remains unclear.

Purpose of the Study:

• To elucidate the mechanism by which BART interacts with and regulates ARL3 activity.
• To investigate the role of BART in the context of ARL3 activation and ciliary function.
• To understand the contribution of the BART-ARL3 interaction to ciliopathies.

Main Methods:

• Biochemical assays to assess ARL3 activation by ARL13B and BART.
• GTP binding and dissociation assays to determine BART's effect on ARL3-GTP stability.
• Live cell imaging to visualize BART localization and co-localization with ARL13B in primary cilia.

Main Results:

• BART acts as a co-GEF with ARL13B, efficiently activating ARL3 at physiological GTP:GDP levels.
• BART binds ARL3-GTP, stabilizing the active G-protein by inhibiting GTP dissociation.
• BART localizes to primary cilia and co-localizes with ARL13B, suggesting a functional role in this compartment.

Conclusions:

• BART functions as a co-GEF for ARL3, working in concert with ARL13B to promote ARL3 activation.
• BART stabilizes active ARL3-GTP, ensuring its availability until recruited by downstream effectors.
• The findings provide a new mechanistic insight into ARL3 regulation and its implications for ciliopathies.