Whatmore J, Morgan C P, Cunningham E, Collison K S, Willison K R, Cockcroft S
Department of Physiology, University College London, U.K.
Biochem J. 1996 Dec 15;320 ( Pt 3)(Pt 3):785-94. doi: 10.1042/bj3200785.
ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.
ADP-核糖基化因子（ARF）是一种小GTP酶，是囊泡形成所必需的，已被鉴定为磷脂酶D（PLD）的激活剂，这意味着PLD定位于细胞内细胞器。HL60细胞用[14C]乙酸预标记72小时，破碎后在线性蔗糖梯度上进行分级分离。通过测量磷脂酰乙醇的产生来评估每个级分中ARF1调节的PLD活性。鉴定出两个活性峰，分别与高尔基体/内质网/颗粒（内膜）和质膜的标志物一致。使用外源性磷脂酰胆碱作为底物对级分进行分析，证实内膜和质膜中存在ARF1依赖性PLD活性，并且还在细胞质中鉴定出额外的活性。在甲酰甲硫氨酰亮氨酰苯丙氨酸刺激的细胞中，通过磷脂酰乙醇形成评估的PLD活性也与质膜和内膜相关。由于ARF1调节的PLD活性需要磷脂酰肌醇4,5-二磷酸（PIP2），因此研究了肌醇脂质和负责脂质磷酸化的激酶的分布。PIP2在质膜中高度富集，而PIP2合成的前体磷脂酰肌醇（PI）和磷脂酰肌醇4-磷酸（PI4P）主要在内膜中发现。PI 4-激酶和PI4P 5-激酶活性的分布证实质膜是PIP2产生的主要部位。然而，内膜具有大量的PI 4-激酶活性和一些PI4P 5-激酶活性，说明PIP2合成的潜力。得出以下结论：（1）ARF1调节的PLD活性定位于内膜和质膜，（2）两个膜区室均有PIP2作为ARF调节的PLD的辅因子发挥作用，（3）在完整细胞中，甲酰甲硫氨酰亮氨酰苯丙氨酸刺激内膜以及质膜上的PLD活性。
