Liu J, Hughes T E, Sessa W C
Molecular Cardiobiology Program and the Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
J Cell Biol. 1997 Jun 30;137(7):1525-35. doi: 10.1083/jcb.137.7.1525.
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.
具有催化活性的内皮型一氧化氮合酶（eNOS）定位于高尔基体复合体以及内皮细胞（EC）的小窝中。由N-肉豆蔻酰化或半胱氨酸棕榈酰化位点突变导致的eNOS定位错误会损害刺激型一氧化氮（NO）的产生，这表明细胞内靶向对于最佳NO产生至关重要。为了研究EC中eNOS靶向的分子决定因素，我们构建了eNOS-绿色荧光蛋白（GFP）嵌合体，以研究其在活细胞和固定细胞中的定位。与野生型（WT）eNOS相比，全长eNOS-GFP融合蛋白与高尔基体标记物甘露糖苷酶II共定位，并保留了催化活性，这表明GFP标签不会干扰eNOS的定位或功能。用与GFP偶联的不同大小的氨基末端融合伴侣进行的实验表明，eNOS的前35个氨基酸足以将GFP靶向到NIH 3T3细胞的高尔基体区域。此外，位于eNOS棕榈酰化位点（Cys-15和-26）之间的独特（Gly-Leu）5重复序列对于其棕榈酰化以及因此的定位是必需的，但对于N-肉豆蔻酰化、膜结合和NOS活性不是必需的。棕榈酰化缺陷型突变体显示出比WT eNOS-GFP更弥散的荧光模式，但仍与细胞内膜相关。生化研究还表明，棕榈酰化缺陷型突变体与膜的结合与WT eNOS一样紧密。N-肉豆蔻酰化位点Gly-2的突变（消除了N-肉豆蔻酰化和棕榈酰化）导致GFP融合蛋白像单独的GFP一样分布在整个细胞中，这与其在生化研究中的主要胞质性质一致。因此，eNOS通过前35个氨基酸，包括N-肉豆蔻酰化和棕榈酰化位点，靶向到NIH 3T3细胞的高尔基体区域，并且其整体膜结合需要N-肉豆蔻酰化而不是半胱氨酸棕榈酰化。这些结果表明脂肪酰化在eNOS的特定区室化中具有新作用，并且很可能对其他双重酰化蛋白而言，对高尔基体复合体也有新作用。
