Kinetic analysis of Arf GAP1 indicates a regulatory role for coatomer
Kinetic analysis of Arf GAP1 indicates a regulatory role for coatomer
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DOI：
10.1074/jbc.m802268200
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发表时间：
2008-08-08
影响因子：
4.8
通讯作者：
Randazzo, Paul A.
中科院分区：
文献类型：
--
作者：
Luo, Ruibai;Randazzo, Paul A.
Arf GAP 是催化与 Arf 结合的 GTP 水解的酶家族。 Arf GAP1 是该家族的成员之一，在高尔基体的膜交通中发挥着关键作用。已经提出了两种不同的膜运输 Arf GAP1 调节模型。在一种模型中，Arf GAP1 在与外壳蛋白的三元复合物中发挥作用，并受到货物蛋白的抑制。在另一个模型中，Arf GAP1 被募集到膜表面，该膜表面具有由于伴随运输囊泡形成而增加的膜曲率所产生的缺陷。在这里，我们使用动力学和突变分析来测试 Arf GAP1 调控模型的预测。我们发现Arf GAP1对Arf1.GTP的亲和力与另一种Arf GAP ASAP1相似，但催化速率约为ASAP1的0.5%。涂层刺激 Arf GAP1 活性；然而，与当前模型预测的不同，涂层异构体影响 K-m 而不是 k(cat) 值。 Arf GAP1 中大多数突变的影响与 ASAP1 中的影响相似。与推测在 ASAP1 中起催化作用的精氨酸对齐的精氨酸突变会消除活性。货物蛋白胞质尾部的肽抑制 Arf GAP1；然而，不相关的 Arf GAP ASAP1 也受到抑制。在我们的实验条件下，脂质双层的曲率对 Arf GAP1 的活性影响很小。我们得出结论，coatomer 是 Arf GAP1 的变构调节剂。讨论了结果与 Arf GAP1 介导的 Arf1 调节的两种模型的相关性。
Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximate to 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K-m and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.