A novel motif at the C-terminus of palmitoyltransferases is essential for Swf1 and Pfa3 function in vivo.

S-acylation (commonly known as palmitoylation) is a widespread post-translational modification that consists of the addition of a lipid molecule to cysteine residues of a protein through a thioester bond. This modification is predominantly mediated by a family of proteins referred to as PATs (palmitoyltransferases). Most PATs are polytopic membrane proteins, with four to six transmembrane domains, a conserved DHHC motif and variable C-and N-terminal regions, that are probably responsible for conferring localization and substrate specificity. There is very little additional information on the structure-function relationship of PATs. Swf1 and Pfa3 are yeast members of the DHHC family of proteins. Swf1 is responsible for the S-acylation of several transmembrane SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) and other integral membrane proteins. Pfa3 is required for the palmitoylation of Vac8, a protein involved in vacuolar fusion. In the present study we describe a novel 16-amino-acid motif present at the cytosolic C-terminus of PATs, that is required for Swf1 and Pfa3 function in vivo. Within this motif, we have identified a single residue in Swf1, Tyr323, as essential for function, and this is correlated with lack of palmitoylation of Tlg1, a SNARE that is a substrate of Swf1. The equivalent mutation in Pfa3 also affects its function. These mutations are the first phenotype-affecting mutations uncovered that do not lie within the DHHC domain, for these or any other PATs. The motif is conserved in 70% of PATs from all eukaryotic organisms analysed, and may have once been present in all PATs. We have named this motif PaCCT ('Palmitoyltransferase Conserved C-Terminus').