Juxta-membrane S-acylation of plant receptor-like kinases is likely fortuitous and does not necessarily impact upon function

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 family we find that S-acylation adjacent to receptor-like kinase domains is common, affecting ~25% of Arabidopsis receptor-like kinases, 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 S-acylation 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 S-acylation 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.

Figure 1

FLS2 C^{830, 831}S expressing plant responses to flg22 stimulation are indistinguishable from wild type (A). MAPK activation in fls2/FLS2pro:FLS2 and fls2/FLS2pro:FLS2 C^830,831S seedlings in response to 100 nM flg22 over time as determined by immunoblot analysis. pMAPK6/pMAPK3 show levels of active form of each MAPK. MAPK6 indicates total levels of MAPK6 as a loading control. Upper shadow band in MAPK6 blot is rubisco bound  non-specifically by secondary antibody. (B). Induction of WRKY40, (C) NHL10 and (D) PHI-1 gene expression after 1 hour treatment with 1 mM flg22 in fls2/FLS2pro:FLS2 and fls2/FLS2pro:FLS2 C^830,831S seedlings as determined by qRT-PCR. (E) Induction of PR1 gene expression after 24 hours treatment with 1 mM flg22 in fls2/FLS2pro:FLS2 and fls2/FLS2pro:FLS2 C^830,831S seedlings as determined by qRT-PCR. Values were calculated using the DDCT method from 3 technical replicates, error bars represent RQ_MIN and RQ_MAX and constitute the acceptable error level for a 95% confidence interval according to Student’s t-test. Asterisks denote statistically significant differences between Col-0 flg22 treated sample and other flg22 treated samples (*P < 0.05). Similar data were obtained over 2 biological repeats (additional data shown in Supplemental Fig. 1). (F) Relative quantification of callose deposition following 12 hours treatment with water or 1 mM flg22. Averages are shown from at least 2 biological repeats, error bars represent SEM and statistically similar groupings (P < 0.05) are denoted by a, b, c as determined by ANOVA and post-hoc Tukey-Kramer test. (G) Average relative seedling mass (flg22 treated/untreated) of seedlings grown in 1 µm flg22 for 10 d (14 d post germination). Data are averages of three independent biological replicates. Error bars show SEM. Asterisks denote statistically significant differences compared with that of the Col-0 control (***P < 0.01) determined by one-way ANOVA and Tukey’s HSD test. Full-length blots are presented in Supplementary Fig. 4.

Figure 2

Receptor-like kinases are S-acylated at sites other than the transmembrane region. (A) Receptor-like kinases show reduced but readily apparent S-acylation following mutation of juxta-TM cysteine residues. S-acylation state of receptor-like kinases was determined using Acyl-biotin exchange (ABE; FLS2 variants expressed in Arabidopsis) or Acyl-RAC (LYK4 and ER variants expressed in N. benthamiana) methods. (B) Truncations of LYK4 and ER indicate that S-acylation occurs at juxta-TM cysteines similarly to FLS2. (C) Receptor-like kinases that do not contain juxta-TM cysteines are also S-acylated. HYD indicates presence (+) or absence (−) of hydroxylamine required for S-acyl group cleavage during the thiopropyl Sepharose 6b capture (Acyl-RAC) or biotinylation step (ABE) step. Thiopropyl Sepharose 6b (Acyl-RAC) or neutravidin (ABE) enriched proteins were eluted and represent S-palmitoylated proteins (EX). Prior to thiopropyl Sepharose 6b (Acyl-RAC) or neutravidin (ABE) capture a sample was removed as an input loading control (LC). Full-length blots are presented in Supplementary Fig. 4.

Figure 3

Receptor-like kinase localisation to the plasma membrane does not require S-acylation. Receptor-like kinase variants fused to EGFP (green) were imaged in Lti6b-mOrange (magenta) expressing plants. Chlorophyll auto-fluorescence is shown in blue. Scale bar represents 10 μm.