THE FIRST 35 AMINO-ACIDS AND FATTY ACYLATION SITES DETERMINE THE MOLECULAR TARGETING OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE INTO THE GOLGI REGION OF CELLS - A GREEN FLUORESCENT PROTEIN STUDY

Catalytically active endothelial nitric oxide synthase (eNOS) is locat ed on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for o ptimal NO production. To investigate the molecular determinants of eNO S targeting in EC, we constructed eNOS-green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The ful l-length eNOS-GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, s uggesting that the GFP tag does not interfere with eNOS localization o r function. Experiments with different size amino-terminal fusion part ners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)(5) repeat located between the palmi toylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoy lation and thus localization, but not for N-myristoylation, membrane a ssociation, and NOS activity. The palmitoylation-deficient mutants dis played a more diffuse fluorescence pattern than did WT eNOS-GFP, but s till were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caus ed the GFP fusion protein to distribute throughout the cell as GFP alo ne, consistent with its primarily cytosolic nature in biochemical stud ies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells vi a the first 35 amino acids, including N-myristoylation and palmitoylat ion sites, and its overall membrane association requires N-myristoylat ion but not cysteine palmitoylation. These results suggest a novel rol e for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex .