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Molecular and Cellular Biology, March 2007, p. 2144-2154, Vol. 27, No. 6
0270-7306/07/$08.00+0     doi:10.1128/MCB.02347-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Activation Status-Coupled Transient S Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase ^,

Department of Plant Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel,¹ Department of Field and Vegetable Crops, Agricultural Research Organization, Newe Ya'ar Reseach Center, P.O. Box 1021, Ramat Yishay 30095, Israel²

Received 17 December 2006/ Accepted 1 January 2007

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His₆-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His₆-GFP-Atrop6^CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His₆-GFP-Atrop6^CAmS¹⁵⁶ in which cysteine¹⁵⁶ was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.

Published ahead of print on 22 January 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

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