Cutting out the fat: A new screen for de-S-acylases in plants

Protein S-acylation is the covalent and reversible attachment of a fatty acid on a protein’s cysteine via a thioester bond and an important posttranslational modification. In eukaryotes, an acylation cycle is governed by the opposing actions of protein S-acyl transferases (PATs) and de-S-acylases, also called acyl-protein thioesterases (APTs), which regulate the localization of many proteins to the plasma membrane, such as the proto-oncogene product Ras in humans. In plants, the modification is less well investigated despite the identification of many putative acylation targets that are involved in developmental processes and stress responses. While many PATs have been identified in Arabidopsis (Batistic, 2012) and their involvement in different biological processes, for example, leaf senescence has been shown, clear Arabidopsis homologs of the mammalian APTs have not been identified (Hemsley, 2020).

In a new publication, Xiaoshi Liu, Min Li, Yang Li, and colleagues (Liu et al., 2021) present a screen for the identification of APTs in Arabidopsis (see Figure). The authors used a recently discovered mammalian de-S-acylase family as a starting point for a new computer-based search to identify 11 homologs in Arabidopsis, which they named ABHD17-like APTs (ABAPTs). Next, they selected five potential target proteins that might serve as deacylation substrates. These five proteins play important roles in plant immunity, and two of them had already been proposed as S-acylation substrates, albeit no biochemical evidence was presented (Takemoto and Jones, 2005; Qi et al., 2014). Liu and colleagues fused these putative protein substrates to GFP, expressed them in protoplasts, and confirmed their expected localization at the plasma membrane. After mutating their predicted acylation sites, the proteins were instead found in the cytoplasm, further confirming that they were acylation targets.

Figure.

An acylation/deacylation cycle governed by PATs and ABAPTs controls the localization of many proteins. Using a new screen, Liu et al. (2021) identify a series of Arabidopsis ABAPTs and some of their protein substrates. Created with BioRender.com.

The authors then set out to investigate the specificity of their identified ABAPTs. They tagged the 11 ABAPTs with GFP and the five protein substrates with RFP and co-expressed all possible combinations in protoplasts to study their localization using confocal microscopy. A shift of a substrate’s localization from the plasma membrane to the cytoplasm upon co-expression with an ABAPT and a matching co-localization indicated deacylation of the substrate by the ABAPT. Using these methods, they successfully discovered new APTs in Arabidopsis and confirmed that the putative substrates undergo an acylation/deacylation cycle. For example, they identified ABAPT8 as a de-S-acylase for RIN4, an essential regulator of plant defense. PBS1, an important protein kinase in plant immunity, was found to be a target of ABAPT11. The authors complemented their microscopy results by demonstrating localization of the acylated and deacylated proteins in different cellular fractions after ultra-centrifugation, and they confirmed the acylated state of their substrates with a so-called biotin-switch assay, in which samples from cell lysates can be analyzed for acylation.

The new screen developed by Liu et al. allows for relatively quick studies of the dynamic regulation of substrate S-acylation in Arabidopsis, and it will be exciting to learn about the targets of the entire set of 11 ABAPTs in the future, and of the TIPSY1 protein (B�rger et al., 2017), which the authors used as a control. Most importantly, the screen could easily be adopted to other plant species, which makes these results an important advancement in the field of plant S-acylation and of plant immunity at large.