MUTAGENESIS OF PALMITOYLATION SITES IN ENDOTHELIAL NITRIC-OXIDE SYNTHASE IDENTIFIES A NOVEL MOTIF FOR DUAL ACYLATION AND SUBCELLULAR TARGETING

The endothelial nitric oxide synthase (ecNOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle, ecNOS undergoes a complex series of covalent modifications, in cluding myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association, Mutagenesis of the myristoylation site , which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergo es partial redistribution to the cell cytosol concomitant with depalmi toylation, To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at hco potentia l palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesi s. Immunoprecipitation of ecNOS mutants following cDNA transfection an d biosynthetic labeling with [H-3]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacem ent of both residues completely eliminated palmitoylation, Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region c ontaining these two cysteine residues is both necessary and sufficient for enzyme palmitoylation, The cysteines thus identified as the palmi toylation sites for ecNOS are separated by an unusual (Gly-Leu)(5) seq uence and appear to define a sequence motif for dual acylation, We ana lyzed the subcellular distribution of ecNOS mutants by differential ul tracentrifugation and found that mutagenesis of the ecNOS palmitoylati on sites markedly reduced membrane association of the enzyme, These re sults document that ecNOS palmitoylation is an important determinant f or the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.