Identification of a mammalian palmitoyltransferase that modifies transmembrane SNARE proteins

CHUMPÉN, S; VALDEZ TAUBAS J

Puerto Madrin

Congreso; XLVI SAIB; 2010

ciedad Argentina de Investigación en Bioquímica y Biología Molecular

Protein palmitoylation or S-acylation is a post-translational modification affecting a large number of proteins. The enzymes responsible for this modification are characterized by the presence of a DHHC-Cysteine Rich Domain. The yeast S. cerevisiae has 7 members of this family in its genome, and there are 23 in mammals. Several transmembrane SNAREs, are palmitoylated both in yeast and mammals. In yeast, this modification is carried out by the Swf1 protein. The mammalian orthologue, has not been identified. Although these proteins display low conservation outside the DHHC domain, sequence analyses point to DHHC4 as a putative candidate. To investigate if DHHC4 is the enzyme responsible for mammalian SNAREs palmitoylation, we generated a shRNA against the DHHC4 sequence, to knock-down the protein expression levels in HEK 293 cells. The efficiency of the knock-down was assessed using specific antibodies against the DHHC4 C-terminus, generated in our laboratory. This antibodies are also being use to study the intracellular localization of this protein. Initially, we used DHHC4 silenced HEK 293 cells, to assess the palmitoylation status of the yeast SNARE Tlg1. We have found that Tlg1 palmitoylation is severely reduced in these cells, suggesting that DHHC4 might indeed be the mammalian SNARE palmitoyltransferase. Several mammalian SNARES are currently being tested in the same manner