Assaying protein palmitoylation in plants

Hemsley, Piers A.; Taylor, L. Follansbee; Grierson, Claire

doi:10.1186/1746-4811-4-2

Cited by 57 publications

(57 citation statements)

References 30 publications

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“…To access palmitoylation, we used a modified version of the acyl-biotinyl switch assay (14,31). In this assay, NEM was added to the protein lysates to block all free sulfhydryl groups.…”

Section: Gfp-ras1 Gfpras1(c203s) Gfp-ras1(c204s) and Gfp-ras1(c203mentioning

confidence: 99%

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

Ferreyra

Ballou

et al. 2009

Eukaryot Cell

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In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.Ras proteins are important determinants of cell morphology, differentiation, and stress response in diverse organisms.

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“…To access palmitoylation, we used a modified version of the acyl-biotinyl switch assay (14,31). In this assay, NEM was added to the protein lysates to block all free sulfhydryl groups.…”

Section: Gfp-ras1 Gfpras1(c203s) Gfp-ras1(c204s) and Gfp-ras1(c203mentioning

confidence: 99%

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

Ferreyra

Ballou

et al. 2009

Eukaryot Cell

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“…1A). To determine whether the Cys-3 and Cys-6 residues of PBS1 are modified through thioester linkage to acyl groups, we assessed protein acylation using a "biotin switch" assay (Hemsley et al, 2008;Hemsley, 2013). This method uses hydroxylamine-induced cleavage of thioester bonds, which results in free sulfhydryl groups that can then be conjugated to a biotin derivative, Biotin-BMCC (see "Materials and Methods").…”

Section: S-acylation Likely Mediates the Pm Localization Of Bothmentioning

confidence: 99%

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent onS-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

et al. 2013

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The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a highthroughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification.

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“…The underestimation of proteins found at the membrane can be partially explained by the presence of proteins that, despite lacking transmembrane domains, establish strong non-covalent interactions with integral membrane proteins or undergo covalent attachment of fatty acids in processes such as S-acylation (like palmitoylation) or N-terminal myristoylation. For instance, in Arabidopsis, 319 proteins are myrisotylated (Podell and Gribskov, 2004), and approximately 600 are S-acylated (Hemsley et al, 2008). Membrane proteins and the different ways by which they bind the phospholipid bilayers offer a vast network of potential interaction mechanisms with varying biophysical properties.…”

Section: Introductionmentioning

confidence: 98%

Co-Immunoprecipitation of Membrane-Bound Receptors

Ávila-Pacheco

Lee

Torii

2015

The Arabidopsis Book

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The study of cell-surface receptor dynamics is critical for understanding how cells sense and respond to changing environments. Therefore, elucidating the mechanisms by which signals are perceived and communicated into the cell is necessary to understand immunity, development, and stress. Challenges in testing interactions of membrane-bound proteins include their dynamic nature, their abundance, and the complex dual environment (lipid/soluble) in which they reside. Co-Immunoprecipitation (Co-IP) of tagged membrane proteins is a widely used approach to test protein-protein interaction in vivo. In this protocol we present a method to perform Co-IP using enriched membrane proteins in isolated microsomal fractions. The different variations of this protocol are highlighted, including recommendations and troubleshooting guides in order to optimize its application. This Co-IP protocol has been developed to test the interaction of receptor-like kinases, their interacting partners, and peptide ligands in stable Arabidopsis thaliana lines, but can be modified to test interactions in transiently expressed proteins in tobacco, and potentially in other plant models, or scaled for large-scale protein-protein interactions at the membrane.

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Assaying protein palmitoylation in plants

Cited by 57 publications

References 30 publications

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent onS-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

Co-Immunoprecipitation of Membrane-Bound Receptors

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