A Cell-Specific, Prenylation-Independent Mechanism Regulates Targeting of Type II RACs

Abstract

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO·GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV 15 , were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV 15 did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.

Functional analysis of barley RAC/ROP G‐protein family members in susceptibility to the powdery mildew fungus

“…The transport of barley type II RAC/ROPs to the PM corresponds with data obtained from Arabidopsis and maize. As previously shown, the membrane-targeting signal in the C-terminal hypervariable region of type II RAC/ROPs does not depend on a functional CAAX-box (Ivanchenko et al, 2000;Lavy et al, 2002). Especially the maize PM-located ZmROP6 exhibits very high similarities with HvROP6, even in the hypervariable region (22 of the last 31 aa are identical).…”

“…Moreover, Arabidopsis type I RAC/ROP proteins, which have different functions (Bischoff et al, 2000;Cheung et al, 2003), are very similar in the insert region (Valster et al, 2000;Winge et al, 1997). The second domain of the RAC/ROP proteins without high similarities is the HVR at the C-terminus, thought to be required for subcellular targeting (Bischoff et al, 2000;Lavy et al, 2002). Within this domain, the basic character of the polybasic region is conserved but not the amino acids themselves.…”

“…The second one is the C-terminal HVR that contains signals targeting the RAC/ROP protein to speci®c membranes. HvRACB and HvRACD contain typical prenylation signals (CAAX-boxes, for cysteine±aliphatic aaaliphatic aa-X) at their C-terminus, whereas the HVRs of all other RAC/ROPs contain additional cysteine residues that might be palmitoylated (Ivanchenko et al, 2000;Lavy et al, 2002). The length of the C-terminal region also distinguishes the two type I RAC/ROPs, HvRACB and HvRACD, from the four type II RAC/ROPs, HvRAC1, HvRAC3, HvROP4 and HvROP6 (Figure 1).…”

“…The activity status of plant RAC/ROP proteins is adjusted most likely by receptor-like kinases, GTPase activating proteins and guanine nucleotide dissociation inhibitors (Baxter-Burrell et al, 2002;Vernoud et al, 2003). RAC/ROPs associate with the plasma membrane (PM) mediated by either prenylation or palmitoylation of cysteine residues in the C-terminal HVR (Ivanchenko et al, 2000;Lavy et al, 2002). RAC/ROPs are involved in actin cytoskeleton remodelling, secondary wall formation, establishing of intracellular Ca 2 gradients, regulation of polar cell growth, production of reactive oxygen intermediates (ROIs) and the modulation of hormone signalling and gene expression (Valster et al, 2000;Yang, 2002).…”

GeBP and GeBP-Like Proteins Are Noncanonical Leucine-Zipper Transcription Factors That Regulate Cytokinin Response in Arabidopsis

“…T-DNA insertion lines gpl1-1 (WiscDsLox391A04), gpl2-2 (SALK_054183), and gpl3-3 (SAIL_885_ B10) were obtained from the Arabidopsis Biological Resource Center (Ohio State University) or the Nottingham Arabidopsis Stock Centre and are in the Col-0 background. Agrobacterium tumefaciens C58 pGV3121 was used for stable transformation of Arabidopsis (Col-0 ecotype) using the floral dip technique (Clough and Bent, 1998) or transient expression in tobacco leaves (Nicotiana benthamiana; Lavy et al, 2002).…”