A proteomic approach identifies many novel palmitoylated proteins in Arabidopsis

Abstract

SummaryS-acylation (palmitoylation) is a poorly understood post-translational modification of proteins involving the addition of acyl lipids to cysteine residues. S-acylation promotes the association of proteins with membranes and influences protein stability, microdomain partitioning, membrane targeting and activation state. No consensus motif for S-acylation exists and it therefore requires empirical identification.Here, we describe a biotin switch isobaric tagging for relative and absolute quantification (iTRAQ)-based method to identify S-acylated proteins from Arabidopsis. We use these data to predict and confirm S-acylation of proteins not in our dataset.We identified c. 600 putative S-acylated proteins affecting diverse cellular processes. These included proteins involved in pathogen perception and response, mitogen-activated protein kinases (MAPKs), leucine-rich repeat receptor-like kinases (LRR-RLKs) and RLK superfamily members, integral membrane transporters, ATPases, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs) and heterotrimeric G-proteins. The prediction of S-acylation of related proteins was demonstrated by the identification and confirmation of S-acylation sites within the SNARE and LRR-RLK families. We showed that S-acylation of the LRR-RLK FLS2 is required for a full response to elicitation by the flagellin derived peptide flg22, but is not required for localization to the plasma membrane.Arabidopsis contains many more S-acylated proteins than previously thought. These data can be used to identify S-acylation sites in related proteins. We also demonstrated that S-acylation is required for full LRR-RLK function.

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning:Arabidopsis h-Type Thioredoxins as a Case Study

“…Our results are in strong agreement with very recent published data showing by an independent targeted proteomic approach that TRXh9 indeed belongs to the pool of identified PALed proteins of Arabidopsis (Hemsley et al, 2013).…”

“…Similar to animals, there are 24 predicted PATs in plants (Hemsley et al, 2005;Batistic, 2012), and it is known that each of them displays a specific intracellular localization to the various membrane compartments of the cells with most proteins localized to the PM. It is remarkable that among the pool of cotranslationally predicted MYRed proteins shown recently to undergo PAL, 97% of them display a Cys at either position 3 and/or 4 (80%) or 5 and 6 for the others (see Supplemental Table 9 in Hemsley et al, 2013), confirming as a result the annotation of predicted PALed proteins in the Arabidopsis myristoylome. In this large-scale analysis, made using a targeted proteomics of PALed proteins of Arabidopsis, Hemsley et al (2013) could also identify TRXh9 among all the aforementioned families of both MYRed and PALed proteins.…”

“…It is remarkable that among the pool of cotranslationally predicted MYRed proteins shown recently to undergo PAL, 97% of them display a Cys at either position 3 and/or 4 (80%) or 5 and 6 for the others (see Supplemental Table 9 in Hemsley et al, 2013), confirming as a result the annotation of predicted PALed proteins in the Arabidopsis myristoylome. In this large-scale analysis, made using a targeted proteomics of PALed proteins of Arabidopsis, Hemsley et al (2013) could also identify TRXh9 among all the aforementioned families of both MYRed and PALed proteins. Our data also support previous studies that identified the PM localization of Arabidopsis TRXh9 and possible cell-to-cell movement of the protein through plasmodesmata (Meng et al, 2010).…”

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus

“…In silico analyses using the GPS-Lipid and CSS-Palm tools (Ren et al, 2008;Xie et al, 2016) predicted the presence of Sacylation sites in several IQD family members (Supplemental Table S1). Experimental support for the lipidation of IQD proteins is provided by a large-scale proteomics data set, which reported S-acylation of IQD32 (Hemsley et al, 2013).…”

Protein S-Acyltransferase 14: A Specific Role for Palmitoylation in Leaf Senescence in Arabidopsis

“…Protein S-acylation, commonly known as S-palmitoylation, is a posttranslational lipid modification in which long chain fatty acids, usually palmitate, attach reversibly to cysteines (Resh, 2006;. S-acylation, often coupled with myristoylation or prenylation, is important for cellular protein sorting, vesicle trafficking, activation state control, protein stability, microdomain partitioning of proteins, and protein complex assembly (Greaves and Chamberlain, 2007;Baekkeskov and Kanaani, 2009;Charollais and Van Der Goot, 2009;Hemsley, 2009;Fukata and Fukata, 2010;Hemsley et al, 2013). Recent large-scale proteomics studies indicate that many proteins are palmitoylated in eukaryotic organisms, with at least 50 occurring in yeast , more than 700 in mammals (Martin and Cravatt, 2009), and more than 500 in Arabidopsis (Arabidopsis thaliana; Hemsley et al, 2013).…”