The Palmitoyltransferase Swf1 Is A Bona Fide Zinc Binding Protein

AYELÉN GONZÁLEZ MONTORO; RODRIGO QUIROGA; JAVIER VALDEZ TAUBAS

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2012

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

S-acylation of proteins is catalysed by a family of palmitoyltransferases characterised 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 localised within the DHHC domain. Homology modelling of the tertiary structure of Swf1 DHHC-CRD shows that it could contain two C3H zinc binding pockets, in a structure formed by three beta hairpins. 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 indicate 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. Finally we show directly that recombinant Swf1 DHHC-CRD domain is able to bind zinc. Conservation and complementation studies suggest that the DHHC domain of palmitoyltransferases which have lost the amino acids that coordinate zinc may still acquire a similar structure by alternative stabilization mechanisms.