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.
Highlights
S-acylation is a post-translational protein modification where fatty acids, such as palmitate or stearate, are covalently linked to specific cysteines through thioester linkages
We previously proposed that loss of AtFLS2 juxta-transmembrane S-acylation in AtFLS2 C830,831S mutants had a mild negative effect on flagellin signalling outputs when expressed as C-terminal mGFP6 fusions at 100–300x normal levels[5]
We have since determined that epitope fusions, in particular mGFP6, can have deleterious and unpredictable effects on AtFLS2 function when AtFLS2 is expressed at physiological levels, calling the biological relevance of these data into question[18]
Summary
S-acylation is a post-translational protein modification where fatty acids, such as palmitate or stearate, are covalently linked to specific cysteines through thioester linkages. We recently identified numerous proteins from Arabidopsis as being S-acylated through a proteomics screen[5] Included in this group of S-acylated proteins were members of the receptor-like kinase (RLK) superfamily. RLKs are one of the largest gene families in Arabidopsis with approximately 440 members[6] and their roles in a wide variety of plant processes have been well-characterised This includes plant growth and development through the brassinosteroid hormone receptor BRI1 and its co-receptor BAK17, and the control of organ formation and cell fate specification through ERECTA8. Based on data from the original proteomics screen, we identified juxta-TM S-acylation sites in the model Arabidopsis thaliana RLK FLS25 These data have subsequently been validated using orthogonal metabolic labelling strategies putting S-acylation of FLS2 at these sites beyond reasonable doubt[16]. We decided to revisit our earlier work in greater depth using untagged forms of AtFLS2 expressed close to native levels to accurately gauge the role and contribution of juxta-TM S-acylation to AtFLS2 function
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