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

Abstract

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').

PROTEIN S-ACYL TRANSFERASE10 Is Critical for Development and Salt Tolerance in Arabidopsis

“…Although PATs of a given species can localize at diverse membrane compartments (Ohno et al, 2006;Greaves and Chamberlain, 2011;Batistic, 2012), little is known about the targeting determinants. Mutation at a palmitoyltransferase conserved C terminus (PaCCT) motif of yeast Pfa3 resulted in its functional loss and mistargeting to the vacuolar lumen (González Montoro et al, 2009). However, the PaCCT motif is not present in Arabidopsis PAT10.…”

The Canonical DHHC Motif Is Not Absolutely Required for the Activity of the Yeast S-acyltransferases Swf1 and Pfa4

“…A recent study on the yeast PAT Akr1 has indicated that the protein can still be acylated in trans when this cysteine is mutated, indicating that other sites for S-acylation are possible (23). Moreover, a recent proteomics study has shown that the human DHHCs 5, 6, and 8 are autoacylated outside of the DHHC-CRD in three cysteines located in the C-terminal region in a CCX [7][8][9][10][11][12][13] C(S/T) motif (24). However, this motif is not present in other human or yeast PATs.…”

“…Outside of this domain, two other short motifs, TTXE and DPG, are also highly conserved (4). Finally, the palmitoyltransferase conserved C-terminal (PaCCT) motif is present in most PATs (7).…”

Protein palmitoylation: Palmitoyltransferases and their specificity

“…Additionally, a diminished function of yeast Pfa3 was observed when PaCCT phenyloalanine 250 was mutated to alanine. 20 Conserved structural motifs are shown in Figure 2.…”

“…19 Most DHHC enzymes also contain a Cterminal palmitoyltransferase conserved C-terminus (PaCCT) motif (Figure 1). 20 The mutation of tyrosine 323 that is located within PaCCT results in the lack of Swf1 palmitoylation activity in vivo. Additionally, a diminished function of yeast Pfa3 was observed when PaCCT phenyloalanine 250 was mutated to alanine.…”