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

Abstract

Catalytically active endothelial nitric oxide synthase (eNOS) is located 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 optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS-green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS-GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners 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 palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS-GFP, but still 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) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.

Figure 2

Schematic illustration of eNOS–GFP fusions (A) and mutants (B). M, myristate; P, palmitate.

Figure 1

eNOS–GFP has the same intracellular localization as WT eNOS and colocalizes with Man II. NIH 3T3 cells transfected with GFP, WT eNOS– GFP, or WT eNOS were visualized in live cells by fluorescence microscopy (A), or colocalized with a Golgi marker, Man II, in fixed cells (B and C). IF, immunofluorescence.

Figure 8

Inhibition of eNOS palmitoylation does not alter its overall membrane association. NIH 3T3 cells were transfected with WT, C15/26S, L2S, C/L2S, or G2A eNOS–GFP constructs, and lysates were separated into cytosolic (C) and membrane (M) and fractions. The GFP proteins were immunoprecipitated with a GFP polyclonal antibody and analyzed by Western blotting with an eNOS mAb.

Figure 3

Targeting of deletion mutants of eNOS-GFP. (A) NIH 3T3 cells were transfected with WT eNOS–GFP or truncated eNOS–GFP constructs, and GFP fluorescence was visualized in live cells by fluorescence microscopy. (B) Fatty acylation of immunoprecipitated eNOS (1–35) GFP and WT eNOS–GFP constructs and relative partitioning of the 1–35 construct between cytosol (C) and membrane (M) fractions (compared to a soluble G2A eNOS [1–73] GFP construct) were determined.

Figure 4

The first 35 amino acids of eNOS constitute a Golgi-targeting signal. (A) eNOS (1–35/74–131) GFP was visualized in live cells (left panel) or colocalized with Man II in fixed cells (center and right panels). (B) eNOS (1–35) G₁₀-GFP was visualized in live cells (left panel) or colocalized with Man II in fixed cells (center and right panels). (C) eNOS (1–35/74– 131) GFP and eNOS (1–35) G₁₀-GFP are myristoylated and palmitoylated. NIH 3T3 cells transfected with GFP or eNOS–GFP constructs were labeled with [³H]myristic acid or [³H]palmitic acid. The GFP proteins were immunoprecipitated with GFP antibodies and analyzed by fluorography. The bands are appropriate sizes from the coding regions.

Figure 5

Lipid modifications influence the subcellular targeting of eNOS–GFP constructs. NIH 3T3 cells were transfected with G2A eNOS–GFP (nonacylated, A) or C15/26S eNOS–GFP (myristoylated but not palmitoylated, B) constructs, and were colocalized in fixed cells with the resident Golgi protein Man II.

Figure 6

Mutation of the leucines between the palmitoylation sites abolishes eNOS palmitoylation. NIH 3T3 cells were transfected with eNOS–GFP constructs, and the cells were labeled with [³H]myristic acid or [³H]palmitic acid. The GFP proteins were immunoprecipitated with GFP antibodies and analyzed by fluorography.

Figure 7

The glycine-leucine repeat is necessary for eNOS intracellular localization. NIH 3T3 cells were transfected with L2S or C/L2S eNOS–GFP constructs, and proteins were colocalized with Man II. Note that the L2S and C/L2S proteins appear to be mislocalized in a similar manner, suggesting that palmitoylation is required for proper Golgi localization.