Juxta-membrane S-acylation of plant receptor-like kinases – fortuitous or functional?

Abstract

S-acylation is a common post-translational modification of membrane protein cysteine residues with many regulatory roles. S-acylation adjacent to transmembrane domains has been described in the literature as affecting diverse protein properties including turnover, trafficking and microdomain partitioning. However, all of these data are derived from mammalian and yeast systems. Here we examine the role of S-acylation adjacent to the transmembrane domain of the plant pathogen perceiving receptor-like kinase FLS2. Surprisingly, S-acylation of FLS2 adjacent to the transmembrane domain is not required for either FLS2 trafficking or signalling function. Expanding this analysis to the wider plant receptor-like kinase superfamily we find that S-acylation adjacent to receptor-like kinase domains is common but poorly conserved between orthologues through evolution. This suggests that S-acylation of receptor-like kinases at this site is likely the result of chance mutation leading to cysteine occurrence. As transmembrane domains followed by cysteine residues are common motifs for Sacylation to occur, and many S-acyl transferases appear to have lax substrate specificity, we propose that many receptor-like kinases are fortuitously S-acylated once chance mutation has introduced a cysteine at this site. Interestingly some receptor-like kinases show conservation of S-acylation sites between orthologues suggesting that S-acylation has come to play a role and has been positively selected for during evolution. The most notable example of this is in the ERECTA-like family where Sacylation of ERECTA adjacent to the transmembrane domain occurs in all ERECTA orthologues but not in the parental ERECTA-like clade. This suggests that ERECTA S-acylation occurred when ERECTA emerged during the evolution of angiosperms and may have contributed to the neo-functionalisation of ERECTA from ERECTA-like proteins.

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signalling

“…These data demonstrate that, in common with other post-translational modifications, S-acylation may affect multiple sites within an RK with differing effects on RK function (e.g. this work and [25]). The position and effect of the S-acylation site identified here at the C-terminus of the FLS2 and EFR kinase domains is highly conserved amongst plant RKs, and is also found in the closely related receptor-like cytoplasmic kinases (RLCKs) that act downstream of activated RKs.…”

“…Altogether, our observations indicate that FLS2 S-acylation stabilises FLS2-BAK1 association and maintains FLS2 in an signalling competent state at the plasma membrane. Discussion FLS2, a prototypical RK, has been previously shown to be S-acylated at a pair of juxtatransmembrane domain cysteines (Cys 830,831), but S-acylation at these sites is apparently dispensable for function [25]. Here we demonstrate that FLS2 is S-acylated at additional cysteine residues (Cys 1132,1135) in a ligand-responsive manner and that this is required for efficient flg22-triggered signalling and resistance to P. syringae DC3000 bacterial infection.…”

S-acylation in plants: an expanding field

“…Initial work on receptor-like kinases suggested that Sacylation at juxta-TM cysteines had a mild positive effect on signalling outputs from the Arabidopsis FLS2 (flagellin sensing 2) flagellin receptor [4], however, recent work indicates that this is likely artefactual due to the use of epitope tags that confound FLS2 function [36] and the use of overexpression constructs. A recent reappraisal of these data using untagged FLS2 constructs driven by the native promoter and expressed at wild type levels indicates that juxta-membrane S-acylation of FLS2 is entirely dispensable for function [37]. This unexpected finding prompted a bioinformatics based assessment of juxta-membrane S-acylation in receptor-like kinases across species.…”

“…Interestingly, following on from investigating juxta-TM S-acylation, this study also found that even when juxta-TM S-acylation was abolished both Erecta and FLS2 remained S-acylated, albeit at lower levels. Testing of a range of other receptor-like kinases that naturally lack juxta-TM sites indicated that they were also S-acylated and suggests that receptorlike kinases have additional S-acylation sites [37]. The location and function of these sites is currently unknown.…”

“…Cell biology studies using mutagenesis are much more common, but the major downside is that while mutagenesis of a cysteine may alter subcellular localisation there is no direct evidence to support S-acylation of the cysteine in question. While proteins that are solely anchored to membranes by S-acylation are amenable to study via microscopy and mutagenesis, if you are dealing with an integral membrane protein, there is no guarantee that mutation of an S-acylation site will affect subcellular distribution [37] and sub-membrane scale changes are unlikely to be visible without specialized quantitative techniques [39].…”