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Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells
Published on: December 10, 2016
Localization of Rheb to the endomembrane is critical for its signaling function
Claudia Buerger1, Ben DeVries, Vuk Stambolic
1Division of Signaling Biology, Ontario Cancer Institute, University Health Network, 610 University Ave, Toronto, Ont., Canada M5G 2M9.
Biochemical and Biophysical Research Communications
|April 25, 2006
Summary
Farnesylation is crucial for Rheb localization to the endomembrane, enabling its interaction with mTOR. This process is vital for Rheb
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rheb is a small GTPase regulating mTOR activity.
- Rheb possesses a C-terminal CaaX box, undergoing farnesylation.
- Ras superfamily proteins share structural and functional similarities.
Purpose of the Study:
- To investigate the role of farnesylation in Rheb's subcellular localization.
- To determine the impact of Rheb localization on mTOR signaling.
- To elucidate the endomembrane's function in Rheb-mTOR complex formation.
Main Methods:
- Live-cell imaging of EGFP-Rheb.
- Analysis of Rheb association with endomembranes (ER, Golgi).
- Assessment of Rheb-mTOR interaction and downstream signaling (p70S6K phosphorylation).
- Pharmacological inhibition of endomembrane trafficking (Brefeldin A).
Main Results:
- Farnesylation directs Rheb to the endomembrane, specifically Golgi-like structures.
- Impaired endomembrane localization of Rheb disrupts mTOR interaction and signaling.
- Brefeldin A treatment blocks Rheb-mediated p70S6K activation, indicating endomembrane platform necessity.
Conclusions:
- Rheb farnesylation is essential for its proper subcellular localization.
- The endomembrane serves as a platform for Rheb-mTOR complex assembly and signaling.
- Targeting Rheb localization offers a potential strategy for modulating mTOR pathway activity.
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