Dual Lipid Modification of Arabidopsis Gg-Subunits Is Required for Efficient Plasma Membrane Targeting 1[C][W][OA] Qin Zeng, Xuejun Wang, and Mark P. Running* Donald Danforth Plant Science Center, St. Louis, Missouri 63132 Posttranslational lipid modifications are important for proper localization of many proteins in eukaryotic cells. However, the functional interrelationships between lipid modification processes in plants remain unclear. Here we demonstrate that the two heterotrimeric G-protein g-subunits from Arabidopsis (Arabidopsis thaliana), AGG1 and AGG2, are prenylated, and AGG2 is S-acylated. In wild type, enhanced yellow fluorescent protein-fused AGG1 and AGG2 are associated with plasma membranes, with AGG1 associated with internal membranes as well. Both can be prenylated by either protein geranylgeranyltransferase I (PGGT-I) or protein farnesyltransferase (PFT). Their membrane localization is intact in mutants lacking PFT activity and largely intact in mutants lacking PGGT-I activity but is disrupted in mutants lacking both PFTand PGGT-I activity. Unlike in mam- mals, Arabidopsis Ggs do not rely on functional Ga for membrane targeting. Mutation of the sixth to last cysteine, the putative S-acylation acceptor site, causes a dramatic change in AGG2 but not AGG1 localization pattern, suggesting S-acylation serves as an important additional signal for AGG2 to be targeted to the plasma membrane. Domain-swapping experiments suggest that a short charged sequence at the AGG2 C terminus contributes to AGG2’s efficient membrane targeting compared to AGG1. Our data show the large degree to which PFT and PGGT-I can compensate for each other in plants and suggest that differential lipid modification plays an important regulatory role in plant protein localization. Heterotrimeric guanine nucleotide-binding proteins (G proteins) are important components of signal trans- duction pathways and are conserved in all eukaryotic organisms examined (Jones, 2002; Jones and Assmann, 2004; Perfus-Barbeoch et al., 2004). Heterotrimeric G proteins consist of three different subunits, Ga,Gb, and Gg.Gb and Gg are tightly associated as a func- tional unit under physiological conditions, while Ga can signal independently or through Gbg. In the human genome, there are a total of 17 Ga genes that encode 23 Ga-subunits, five Gb genes, and at least 12 Gg genes (Cabrera-Vera et al., 2003), while in the Arabidopsis (Arabidopsis thaliana) genome, only one canonical Ga (GPA1, At2G26300), one Gb (AGB1, At4G34460), and two Gg genes (AGG1, At3g63420 and AGG2, At3g22942) have been identified (Ma et al., 1990; Mason and Botella, 2000; Lease et al., 2001; Mason and Botella, 2001). The same number of G-protein components exists in the rice (Oryza sativa) genome (Kato et al., 2004). Studies of plant Ga and Gb mutants have implicated G proteins in a wide range of develop- mental and phytohormone-mediated cellular processes (Jones, 2002; Jones and Assmann, 2004; Perfus-Barbeoch et al., 2004), including cell division (Ullah et al., 2001, 2003; Warpeha et al., 2006), leaf shape, internode elon- gation (Ueguchi-Tanaka et al., 2000), seed germination (Ullah et al., 2002), stomata function (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2004; Mishra et al., 2006), and pathogen susceptibility (Suharsono et al., 2002; Trusov et al., 2006). Arabidopsis gpa1 and agb1 mutants share some common features, such as rounder rosette leaves, but, consistent with independent roles in par- ticular signaling events have many distinct pheno- types. agb1 mutants produces excessive lateral roots and are more sensitive to auxin promotion of lateral roots, while gpa1 forms fewer lateral roots and is less sensitive to auxin (Ullah et al., 2003). agb1 mutants are more hypersensitive to abscisic acid (ABA) than gpa1 (Pandey et al., 2006), and GPA1 and AGB1 play oppo- site roles in regulating cell proliferation in roots (Chen et al., 2006). Critical to their signaling function is the localization of G proteins to the cytoplasmic face of plasma mem- brane in animal cells (Casey, 1995). This subcellular localization is often facilitated by lipid modifications of the Ga- and Gg-subunits. In mammalian and yeast (Saccharomyces cerevisiae) cells, Ga is usually modified by attachment of a 14-carbon myristate to an N-terminal Gly via an amide bond and/or by linking a 16-carbon palmitate to a nearby Cys through a thioester bond (Milligan et al., 1995). Gg is usually modified by prenylation, attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid via a thioether 1 This work was supported by the National Science Foundation (grant no. IOB–0344261 to M.P.R.). * Corresponding author; e-mail mrunning@danforthcenter.org; fax 314–587–1741. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mark P. Running (mrunning@danforthcenter.org). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a sub- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093583 Plant Physiology, March 2007, Vol. 143, pp. 1119–1131, www.plantphysiol.org Ó 2007 American Society of Plant Biologists 1119 www.plant.org on November 16, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. www.plant.org on November 16, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. www.plant.org on November 16, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. www.plant.org on November 16, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. www.plant.org on November 16, 2015 - Published by www.plantphysiol.org Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved.