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Updated: Jan 31, 2026

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Published on: November 11, 2018

Rab5 GTPases are required for optimal TORC2 function.

Melissa N Locke¹, Jeremy Thorner²

¹Division of Biochemistry, Biophysics, and Structural Biology and Division of Cell and Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA.

The Journal of Cell Biology

|December 23, 2018

Summary

This summary is machine-generated.

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Target of rapamycin complex-2 (TORC2) signaling, crucial for cell membrane stability, is sustained by a positive feedback loop involving Ypk1 kinase and Muk1. This circuit ensures robust TORC2-Ypk1 pathway activation.

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Target of rapamycin complex-2 (TORC2) regulates plasma membrane homeostasis.
Protein kinase Ypk1 is a key downstream effector of TORC2 in budding yeast.

Purpose of the Study:

To investigate the role of Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), as a Ypk1 target.
To elucidate the regulatory circuit sustaining TORC2-Ypk1 signaling.

Main Methods:

Yeast genetics
Co-immunoprecipitation
In vitro kinase assays
Phosphorylation site mapping

Main Results:

Ypk1 phosphorylates and activates Muk1.

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Last Updated: Jan 31, 2026

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

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Published on: November 11, 2018

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

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Disruptions in Rab5 GTPases or their regulators impair TORC2-mediated Ypk1 activation.

TORC2 interacts with Rab5 GTPase Vps21.

TORC2 activity is dependent on Rab5 GTPases.

Conclusions:

TORC2-dependent Ypk1 activation of Muk1 forms a positive feedback loop.
This circuit reinforces TORC2-Ypk1 signaling for sustained pathway activity.