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Related Experiment Video

Updated: Jul 16, 2026

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Published on: March 9, 2012

Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking.

G Eitzen¹, N Thorngren, W Wickner

¹Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.

The EMBO Journal

|October 13, 2001

Summary

Rho GTPases, including Rho1p and Cdc42p, directly regulate vacuole fusion docking. Inhibiting these proteins blocks fusion, highlighting their sequential role in this essential cellular process.

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Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Rho GTPases are key regulators of cell polarity and cytoskeletal dynamics.
Recent studies suggest their involvement in membrane trafficking processes like endo- and exocytosis.

Purpose of the Study:

To investigate the specific role of Rho GTPases, particularly Rho1p and Cdc42p, in homotypic vacuole fusion.
To elucidate the stage at which these GTPases act during the vacuole fusion process.

Main Methods:

Utilized yeast strains with temperature-sensitive alleles of Rho1p and Cdc42p to assess fusion.
Employed Rho GDP dissociation inhibitor (Rdi1p) to extract Rho GTPases and study its effect on vacuole fusion.
Investigated the impact of inhibiting priming and tethering steps on Rho GTPase function.
Assessed the sensitivity of fusion to Rdi1p after docking blockade with BAPTA.

Main Results:

Vacuoles from strains with temperature-sensitive Rho1p or Cdc42p alleles showed thermolabile fusion.
Rdi1p-mediated extraction of Rho1p and Cdc42p from vacuole membranes inhibited fusion.
Rho GTPases function after priming and Ypt7p-dependent tethering but before BAPTA-sensitive docking completion.
Subsequent membrane fusion steps remained sensitive to GTPgammaS even after removal of Ypt7p, Rho1p, and Cdc42p.

Conclusions:

Rho1p and Cdc42p play a direct and sequential role in mediating the docking stage of homotypic vacuole fusion.
These Rho GTPases are essential for the proper execution of vacuole docking, acting in a defined order with other factors like Ypt7p.