ENDOTHELIAL NITRIC-OXIDE SYNTHASE MEMBRANE TARGETING - EVIDENCE AGAINST INVOLVEMENT OF A SPECIFIC MYRISTATE RECEPTOR

The endothelial isoform of nitric oxide synthase (ecNOS) is targeted t o the particulate subcellular fraction by means of N-terminal myristoy lation. However, the association of ecNOS with the particulate subcell ular fraction appears to be dynamically regulated, in that agonist tre atment of endothelial cells induces translocation of the enzyme from m embrane to cytosol (Michel, T., Li, G., and Busconi, L. (1993) Proc. N atl, Acad. Sci. U. S. A. 90, 6252-6255). cDNA encoding wild-type and m yristoylation-deficient mutant (myr-()) ecNOS was transcribed and tran slated in vitro, and we found that the recombinant wild-type but not t he myr(-) mutant protein undergoes myristoylation and is able to assoc iate with biological membranes prepared from diverse cell sources. Tre atment of these cell membranes with heat or with trypsin did not affec t their ability subsequently to serve as acceptor membranes for the wi ld-type recombinant enzyme. The wild-type ecNOS, but not the myr(-) mu tant, is able to form stable associations with phospholipid liposomes. We also explored the possibility that a polybasic domain within the e cNOS protein might serve as a secondary structural determinant for ecN OS membrane association and constructed truncation mutants that flank a polybasic domain present in the ecNOS. These truncation mutants, tra nscribed and translated in vitro or transfected into COS-7 cells, unde rgo myristoylation and are able to associate with biological membranes in a fashion indistinguishable from the wild type ecNOS. Taken togeth er, these results indicate that ecNOS binding to biological membranes is dependent upon interactions of the N-terminal myristoyl moiety of e cNOS with lipid components of the membrane, and this association does not require a specific membrane protein functioning as a myristate rec eptor nor the presence of a polybasic domain within the ecNOS.