Mutational Analysis of the Palmitoyltranferase Swf1

AYELEN GONZALEZ MONTORO; RODRIGO QUIROGA; JAVIER VALDEZ TAUBAS

OXFORD

Simposio; Regulation of protein trafficking and function by palmitoylation; 2012

The Biochemichal Society, UK

S-acylation of proteins is catalysed by a family of enzymes characterized by the DHHC cysteine rich domain. Swf1 is a member of this family, responsible for the modification of SNAREs and glycosyltransferases in yeast. We carried out a random mutagenesis assay designed to uncover essential amino acids in Swf1 and identified 22 novel loss-of-function mutations, most of which are localized within the DHHC domain. Homology modelling of the tertiary structure of Swf1 DHHC-CRD shows that it could contain two C3H zinc binding pockets. The screen revealed that mutation of each of the eight amino acids predicted to be involved in zinc coordination results in inactive Swf1. All of these mutations render Swf1 less stable, suggesting a structural role for Swf1 zinc fingers. Sequence conservation studies of the amino acids that form each zinc binding pocket suggest that upon mutation of one of them the negative selection on the others is lost, in agreement with a function which requires all four of them. It has been proposed that the S-acylation reaction occurs through an acyl-enzyme covalent intermediate that involves the cysteine in the DHHC motif. We have found that a Swf1 mutant in which this cysteine has been replaced for an arginine is partially active. Alleles of PFA4 mutated in this cysteine are also able to partially complement for the deletion of the wt gene when overexpressed.This data suggests that this invariant cysteine may not be absolutely required for the S-acylation reaction.