CONICET | Buscador de Institutos y Recursos Humanos

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists in the thioestherification of cysteine residues with long chain fatty acids, mostly palmitic. This modification is catalyzed by a family of palmitoyltransferases (PATs), characterized by the presence of a 50-residues long cysteine-rich domain (DHHC-CRD). Basic information regarding structure-function relationships of these proteins and the mechanism of protein palmitoylation is lacking. We carried out a random mutagenesis assay designed to uncover essential aminoacids in Swf1, a yeast PAT responsible for the palmitoylation of SNARE proteins and glycosyltransferases. We identified 20 loss-of-function mutations, most of which are localized within the DHHC domain. Homology modeling of the tertiary structure of the Swf1 DHHC-CRD shows that it could contain two zinc binding pockets of the C3H type. All aminoacids predicted to be involved in zinc coordination, were found in the screen as aminoacids that result in inactive Swf1 when mutated. All of these mutations destabilize the protein, suggesting a structural role for Swf1 zinc fingers. Finally, sequence conservation studies of the aminoacids that form each zinc binding pocket, suggest that upon mutation of one of them, the selective pressure to keep the others is lost, in agreement with a shared function which requires all four of them. It has been proposed that the palmitoylation reaction occurs through a palmitoyl-PAT covalent intermediate that involves the conserved cysteine in the DHHC motif. In this screen we have also isolated a partial loss-of-function allele (Swf1 DHHR) which revealed that the invariant cysteine in the DHHC motif, is not essential for the palmitoylation reaction, suggesting an alternative mechanis

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