S-acylation of transmembrane proteins by the yeast Palmitoyltransferase Swf1.

MARIA AYELÉN GONZÁLEZ MONTORO; JAVIER VALDEZ TAUBAS

Maldonado

Simposio; Thiol metabolism and redox regulation of cellular functions; 2011

Instituto Pasteur Uruguay

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with long chain fatty acids, mostly palmitic acid. This modification is catalyzed by a family of palmitoyltransferases (PATs) characterized by the presence of a 50-residue long cysteine-rich domain (DHHC-CRD domain). Basic information regarding structure-function relationships of these proteins and the mechanism of protein palmitoylation is lacking.Yeast has 7 members of the family and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. However, the specificity of PATs is not yet fully understood, since several yeast PATs seem to have overlapping specificity. We showed that Swf1 is highly specific for its substrates, and that there are structural requirements for the modification of Tlg1, a SNARE protein that is modified by Swf1.It has been proposed that the palmitoylation reaction occurs through a palmitoyl-PAT covalent intermediate that involves the conserved cysteine in the DHHC motif. We carried out a random mutagenesis assay designed to uncover essential aminoacids in Swf1. We identified novel loss-of-function mutations, mostly within the DHHC domain. We also isolated some partial loss-of-function alleles that revealed that the conserved cysteine in the DHHC motif is not essential for the palmitoylation reaction, suggesting an alternative reaction mechanism. Besides, mutation of all cysteines in the DHHC-CRD domain result in inactive Swf1, suggesting either a structural role necessary for the function of the domain or a transfer mechanism in which several cysteines are involved.