Tel Aviv 69978, IsraelArabidopsis thaliana calcineurin B-like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-toplasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12-amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca 2þ -decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.

ROP/RAC GTPases are master regulators of cell polarity in plants, implicated in the regulation of diverse signaling cascades including cytoskeleton organization, vesicle trafficking, and Ca(2+) gradients [1-8]. The involvement of ROPs in differentiation processes is yet unknown. Here we show the identification of a novel ROP/RAC effector, designated interactor of constitutive active ROPs 1 (ICR1), that interacts with GTP-bound ROPs. ICR1 knockdown or silencing leads to cell deformation and loss of root stem-cell population. Ectopic expression of ICR1 phenocopies activated ROPs, inducing cell deformation of leaf-epidermis-pavement and root-hair cells [3, 5, 6, 9]. ICR1 is comprised of coiled-coil domains and forms complexes with itself and the exocyst vesicle-tethering complex subunit SEC3 [10-13]. The ICR1-SEC3 complexes can interact with ROPs in vivo. Plants overexpressing a ROP- and SEC3-noninteracting ICR1 mutant have a wild-type phenotype. Taken together, our results show that ICR1 is a scaffold-mediating formation of protein complexes that are required for cell polarity, linking ROP/RAC GTPases with vesicle trafficking and differentiation.

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Membrane attachment of almost all members of the Ras superfamily of small GTP-binding proteins depends on isoprenylation of C-terminal cysteine residues and concomitant proteolysis and carboxy methylation (6-9, 15, 26). Prenylation involves the covalent attachment via a thioether linkage of either farnesylpyrophosphate or geranylgeranylpyrophosphate isoprenoid intermediates. The prenylated cysteines are part of a conserved C-terminal sequence motif designated the CaaX box or double-cysteine motifs in Rab proteins (24). H-Ras, N-Ras, and several Rho proteins are S acylated, as well as prenylated (15). S acylation, more commonly referred to as palmitoylation, involves the attachment of palmitate (C 16:0 ) or other saturated lipids to cysteine residues through a reversible thioester linkage (37).Because of its reversibility, S acylation has attracted much attention as a mechanism modulating signaling by regulation of plasma membrane (PM) localization, attraction to lipid rafts, and protein-protein interactions. Acylated (palmitoylated) proteins tend to partition into anionic detergent-resistant membrane (DRM) domains, suggesting their localization in lipid rafts, while prenylated proteins show little affinity for DRMs (23, 25, 38). Thus, transient S acylation could provide a means to spatially separate proteins. Acylation-deacylation cycles promote transport of H-Ras and N-Ras proteins from the Golgi apparatus to the PM and vice versa (34). Activated H-Ras and K-RasB are excluded from lipid rafts, while the inactive form of H-Ras, but not of K-RasB, accumulates in lipid rafts (30-32).Plant ROPs or RACS are Rac-related GTPases (5) and the only known plant Ras superfamily proteins involved in signaling. ROPs regulate multiple signaling pathways involved in growth, development, and response to pathogens, organization of the actin cytoskeleton, Ca 2ϩ , phosphoinositide, and reactive oxygen species signaling (29). In Arabidopsis and other plants, ROPs constitute multimember protein families. On the basis of differences in gene structure and sequences of their Cterminal hypervariable domains, the plant ROPs were subdivided into two major subgroups designated type I and type II (40). Type I ROPs terminate with a conserved CaaL box motif and a proximal polybasic domain, and it was suggested that they likely undergo prenylation.

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gg proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.

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.

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