Different ARF Domains Are Required for the Activation of Cholera Toxin and Phospholipase D (*)
Different ARF Domains Are Required for the Activation of Cholera Toxin and Phospholipase D (*)
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霍乱毒素和磷脂酶 D 的激活需要不同的 ARF 结构域 (*)
DOI：
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发表时间：
1995
影响因子：
4.8
通讯作者：
M. Vaughan
中科院分区：
文献类型：
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作者：
Gui;W. Patton;F. Lee;M. Liyanage;Joong;S. Rhee;J. Moss;M. Vaughan
adp核糖基化因子（ARFs），最初被描述为霍乱毒素adp核糖基转移酶活性的激活剂，调节细胞内囊泡膜运输并刺激磷脂酶D （PLD）异构体。arf样蛋白（ARL）在结构上与arf相关，但不激活霍乱毒素，对PLD的影响相对较小。利用聚合酶链反应克隆技术，鉴定出一个新的人类ARL基因hARL1，该基因与hARF1具有57%的氨基酸同源性。为了确定霍乱毒素A亚基和PLD的激活是否与不同的结构元件有关，我们通过切换ARF1和ARL1的氨基末端73个氨基酸构建了嵌合蛋白。重组蛋白rL73/F以ARL1的氨基末端73个氨基酸取代ARF1的氨基末端73个氨基酸，能激活霍乱毒素的A亚基，而以ARF1的氨基末端73个氨基酸取代ARL1的氨基末端73个氨基酸的重组蛋白rF73/L则无活性。两种嵌合蛋白对PLD活性的影响完全相反。rF73/L蛋白激活PLD的效果与rARF1相同，而rL73/F蛋白仅轻微激活PLD。结果表明，ARF1的氨基末端区域对其作为霍乱毒素gtp依赖性激活剂的作用并不重要，而它是激活假定的效应酶PLD所必需的。
ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.