Nadav Sorek

A Novel ROP/RAC Effector Links Cell Polarity, Root-Meristem Maintenance, and Vesicle Trafficking

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.

Dual Fatty Acyl Modification Determines the Localization and Plasma Membrane Targeting of CBL/CIPK Ca2+ Signaling Complexes in Arabidopsis

Arabidopsis 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-to-plasma 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 Ca2+-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.

Regulation of membrane trafficking, cytoskeleton dynamics, and cell polarity by ROP/RAC GTPases.

Rho of plants (ROP) proteins, also known as RAC proteins, are Rho-related GTPases that function as molecular switches in a multitude of signaling cascades involved in the regulation of the actin and microtubule cytoskeleton, of vesicle trafficking, and of plant responses to hormones, stresses, or

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

ABSTRACT 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-Atrop6CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS 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-Atrop6CAmS156 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.

Protein lipid modifications in signaling and subcellular targeting

Classically perceived as means for recruiting proteins to the membranes, protein lipid modifications are known today to play diverse roles in subcellular targeting, protein-protein interactions and signaling. This review focuses on three protein lipid modifications: prenylation, S-acylation and N-myristoylation and attempts to provide an up-to-date view of their function by focusing on several model proteins.

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

Summary Rho GTPases are master regulators of cell polarity [1]. For their function, Rhos must associate with discrete plasma membrane domains [2]. Rho of Plants (ROPs) or RACs comprise a single family [3–5]. Prenylation and S -acylation of hypervariable domain cysteines of Ras and Rho GTPases are required for their function [6–11]; 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 [12]. 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 [13] 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.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.

Differential Effects of Prenylation and S-Acylation on Type I and II ROPS Membrane Interaction and Function

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ 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.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Background: The COBRA gene is highly coexpressed with cellulose synthase genes, but its function remains unclear. Results: COBRA localizes at the plasma membrane and binds glucan chains. NMR studies indicate structural defects in cellulose in the mutant despite normal polymerization rate. Conclusion: COBRA functions downstream of cellulose biosynthesis. Significance: This work suggests that alignment of glucan chains into cellulose fibrils is facilitated by one or more proteins. Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.”

Analysis of protein S -acylation by gas chromatography–coupled mass spectrometry using purified proteins

S-acylation, also known as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. Owing to its reversibility, S-acylation is important in regulation of diverse signaling cascades, including Ras-associated cancers in mammals, stress response and metabolic regulation. Here we describe a simple protocol for analysis of protein S-acylation using gas chromatography–coupled mass spectrometry. Analysis can be carried out with as little as 1 μg of purified protein and allows chemical identification and, potentially, quantification of the acyl moieties. The method is based on cleavage of the fatty acids from proteins by hydrogenation with platinum (IV) oxide. This causes an acid transesterification of the acyl groups, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1

Significance The cobra mutants of Arabidopsis, such as cob-6, have impaired growth associated with a defect in cellulose synthesis. Mutations in MEDIATOR16 (MED16) reduce the number of misregulated genes in cob-6 mutants and suppress the phenotypes. This observation implicates MED16 in transcriptional responses to cell wall defects. Ectopic expression of two pectin methylesterase inhibitors (PMEIs) identified in a suppressor screen partially suppressed the growth defect in the cob-6 mutant. The results confirm that the PMEIs have significant in vivo activity, and provide evidence that pectin esterification can modulate cell wall properties. We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose.