Structure-function analysis of yeast S-acyltransferases

AYELEN GONZALEZ MONTORO; RODRIGO QUIROGA; JAVIER VALDEZ TAUBAS

Saxtons River, Vermont,

Conferencia; FASEB Science Research Conference ?Protein Lipidation, Signaling, and Membrane Domains; 2015

FASEB Federation of American Societies for Experimental Biology

STRUCTURE-FUNCTION ANALYSES OF YEAST S-ACYLTRANSFERASESNumerous proteins are post-translationally modified by the addition of a lipid molecule on a cysteine residue by a thioester bond. This S-acylation, also commonly known as palmitoylation, is involved in numerous biological processes of great importance, as the visual cycle, signal transduction and synaptic transmission. PATs are characterized by the presence of a 50 amino acid domain called the DHHC domain. A mutational analysis of the DHHC-CRD domain of Swf1, the PAT for transmembrane SNARE proteins in yeast allowed us to propose a model for the structure of Swf1 DHHC-CRD, in which a role in zinc coordination is assigned to specific cysteines and histidines. Within this domain, the DHHC motif is highly conserved and it has been proposed that the palmitoylation reaction occurs through a palmitoyl-PAT covalent intermediate that involves the conserved cysteine in this motif. Mutation of this cysteine results in lack of function for several PATs, and DHHA or DHHS mutants are regularly used as catalytically inactive controls. In a genetic screen to isolate loss-of-function mutations in the yeast PAT Swf1, we isolated an allele encoding a Swf1 DHHR mutant. Overexpression of this mutant is able to partially complement a swf1∆ strain and to acylate the Swf1 substrates Tlg1, Syn8 and Snc1. Overexpression of the palmitoyltransferase Pfa4, DHHA or DHHR mutants, also results in palmitoylation of its substrate Chs3. We also investigated the role of the first histidine of the DHHC motif. A Swf1 DQHC mutant is also partially active, but a DQHR is not. Finally, we show that Swf1 substrates are differentially modified by both DHHR and DQHC Swf1 mutants. We propose that in the absence of the canonical mechanism, alternative suboptimal mechanisms take place that are more dependent on the reactivity of the acceptor protein.