An S-Acylation Switch of Conserved G Domain Cysteines Is Required for Polarity Signaling by ROP GTPases

Abstract

Rho GTPases are master regulators of cell polarity. For their function, Rhos must associate with discrete plasma membrane domains. Rho of Plants (ROPs) or RACs comprise a single family. Prenylation and S-acylation of hypervariable domain cysteines of Ras and Rho GTPases are required for their function; however, lipid modifications in the G domain have never been reported. Reversible S-acylation involves the attachment of palmitate (C16:0) or other saturated lipids to cysteines through a thioester linkage and was implicated in the regulation of signaling. Here we show that transient S-acylation of Arabidopsis AtROP6 takes place on two conserved G domain cysteine residues, C21 and C156. C21 is relatively exposed and is accessible for modification, but C156 is not, implying that its S-acylation involves a conformational change. Fluorescence recovery after photobleaching beam-size analysis shows that S-acylation of AtROP6 regulates its membrane-association dynamics, and detergent-solubilization studies indicate that it regulates AtROP6 association with lipid rafts. Site-specific acylation-deficient AtROP6 mutants can bind and hydrolyze GTP but display compromised effects on polar cell growth, endocytic uptake of the tracer dye FM4-64, and distribution of reactive oxygen species. These data reveal an S-acylation switch that regulates Rho signaling.

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…This could be because ROP10CA and ROP10DN are possibly localized at different membrane microdomains in the PM. In fact, GTP-bound type I ROP6 of Arabidopsis is partitioned exclusively to lipid rafts by S-acylation to stabilize the protein's interaction with the PM (Sorek et al, 2007(Sorek et al, , 2010. Similarly, the activated type II ROP11 (RAC10) of Arabidopsis, one of the closest homologs of ROP10, was detected in lipid rafts (Bloch et al, 2005).…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Palmitoylation plays a key role in targeting proteins to PM microdomains (Levental et al, 2010). Arabidopsis ROP6, a type I ROP possessing a CaaL box motif, is primarily geranylgeranylated and, when activated, accumulates in DRMs with transient palmitoylation (Sorek et al, 2007(Sorek et al, , 2010. In addition, mammalian Rac1 is palmitoylated at C178, a modification that is essential for targeting to rafts and actin cytoskeleton remodeling (NavarroLérida et al, 2012), and present in not only rafts but also non-rafts, where Rac1 is predominantly inactivated (Moissoglu et al, 2014).…”

The Microtubule-Associated Protein MAP18 Affects ROP2 GTPase Activity during Root Hair Growth

“…Under physiological conditions, the interaction between RhoGDI and the RHO family of proteins was shown to depend on geranylgeranylation of the GTPase (Hoffman et al, 2000;Grizot et al, 2001;Garcia-Mata et al, 2011). Since recombinant ROPs expressed in E. coli are not geranylgeranylated (Sorek et al, 2010), it was important to establish that RhoGDI1 interacts with geranylgeranylated ROP2 purified from plants. To this end, we prepared protein extracts from GFP-ROP2 seedlings and examined whether GFP-ROP2 could be pulled down by GST-AtRhoGDI1/SCN1.…”

“…Previous studies have shown that several ROPs can affect root hair growth (Molendijk et al, 2001;Jones et al, 2002;Bloch et al, 2005;Sorek et al, 2010). ROP2, ROP4, and ROP6 were shown to accumulate at the root hair initiation site (Molendijk et al, 2001;Jones et al, 2002;Payne and Grierson, 2009).…”

“…Changes in intracellular Ca 2+ levels have been suggested to regulate actin dynamics (Molendijk et al, 2001;Jones et al, 2002Jones et al, , 2007Carol et al, 2005;Takeda et al, 2008). ROP11 and ROP6 also were shown to inhibit endocytosis in root hairs in an actinindependent manner (Bloch et al, 2005;Sorek et al, 2010).…”