ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I
ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I
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DOI：
10.1042/0264-6021:3410185
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发表时间：
1999-07-01
影响因子：
4.1
通讯作者：
Cockcroft, S
中科院分区：
文献类型：
--
作者：
Jones, DH;Bax, B;Cockcroft, S
小的gtp结合蛋白adp核糖基化因子（ARF1）对高尔基体形成涂层囊泡至关重要，也是磷脂酶D （PLD）的激活剂。此外，arf1调控的PLD是导致分泌的信号转导通路的一部分。在这项研究中，我们测试了ARF1的替代和缺失突变体激活PLD的能力，这些突变体将ARF1的PLD效应区定位到α 2螺旋、β 2链的一部分和n端螺旋及其随后的环。ARF突变体激活PLD的能力增加或减少，在刺激HL60细胞分泌能力的测试中显示出相似的特征。ARF1缺失了n端17个氨基酸残基（Ndell7），不支持PLD活性或分泌，也不抑制野生型肉豆荚酰基化ARF1 （myrARF1）的活性。相比之下，Ndell7有效地与野生型myrARF1竞争，以阻止涂层与膜的结合，这似乎定义了Ndell7的结构作用，因为它可以结合细胞质中的高分子质量复合物。此外，乙醇对膜吸收涂层没有影响。我们得出结论，arf调节的PLD的功能是在导致溶酶体颗粒分泌的信号转导途径中，而不是作为arf1介导的涂层结合的必要组成部分。
The small GTP-binding protein, ADP-ribosylation factor (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD, These map the PLD effector region of ARF1 to the alpha 2 helix, part of the beta 2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndell7), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndell7 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes, This appears to define a structural role for Ndell7, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding.