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Molecular and Cellular Biology, May 2004, p. 3815-3826, Vol. 24, No. 9
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.9.3815-3826.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The GTPase-Activating Enzyme Gyp1p Is Required for Recycling of Internalized Membrane Material by Inactivation of the Rab/Ypt GTPase Ypt1p

Céline Lafourcade,1 Jean-Marc Galan,2 Yvonne Gloor,1,{dagger} Rosine Haguenauer-Tsapis,2 and Matthias Peter1*

Swiss Federal Institute of Technology Zurich (ETH), Institute of Biochemistry, ETH Hoenggerberg, 8093 Zurich, Switzerland,1 Institut Jacques Monod-CNRS, 75251 Paris Cedex 05, France2

Received 8 September 2003/ Returned for modification 20 October 2003/ Accepted 25 January 2004

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1{Delta} cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.

* Corresponding author. Mailing address: Swiss Federal Institute of Technology Zurich (ETH), Institute of Biochemistry, ETH Hoenggerberg HPM G 6.2, 8093 Zurich, Switzerland. Phone: 41 1 632-3134. Fax: 41 1 632-1269. E-mail: matthias.peter{at}bc.biol.ethz.ch.

{dagger} Present address: Max Planck Institute for Molecular Cell Biology and Genetics, Dresden D-01307, Germany.

Molecular and Cellular Biology, May 2004, p. 3815-3826, Vol. 24, No. 9
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.9.3815-3826.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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