Arabidopsis GHL1 is an orthologue of Hedgehog acyl transferase but likely catalyses GPI-anchor remodelling rather than peptide acylation.

The GUP1/HHAT family of MBOAT proteins have been implicated in GPI-anchor acyl-chain remodelling in fungi and secreted peptide acylation in eumetazoans, but whether these activities are distinct or GUP1/HHAT proteins are bifunctional has not been addressed. We show that the GUP1/HHAT family form a distinct orthologous clade within eukaryotes with structural homology, suggesting a single evolutionary event for their origin and common mode of action. Arabidopsis plants homozygous for loss of GUP1/HHAT-like activity cannot be recovered suggesting that loss is lethal, and further examination suggests that there are severe effects on transmission through the male gamete. Recent work suggests that rice GUP1-like BC16 is a GPI-anchor acyl-chain remodelase, but a potential role for non-eumetazoan GUP1 and HHAT-like proteins in secreted peptide acylation has not been assessed. By reconstituting HHAT peptide acyl transferase activity towards Hedgehog-like peptides in plants we demonstrate that Arabidopsis GUP1/HHAT-like proteins likely do not possess appreciable HHAT-like peptide acyltransferase activity. However, through this work we provide a novel means for cell surface display of proteins in eukaryotic systems, demonstrating that the minimal acyl-acceptor peptide sequence of Hedgehog morphogens, when expressed alongside HHAT, allows for immobilisation of proteins in the outer leaflet of the plasma membrane via their N-terminus, rather than the C-terminus as is the case for traditional GPI-anchor mediated cell surface display.