Tsai S C, Adamik R, Moss J, Vaughan M
Pulmonary/Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):305-9. doi: 10.1073/pnas.93.1.305.
ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.
ADP-核糖基化因子（ARFs）是20 kDa的鸟嘌呤核苷酸结合蛋白，在结合GTP的状态下具有活性，而结合GDP时则无活性。ARF-GTP在几个细胞区室的囊泡运输中起关键作用。鸟嘌呤核苷酸交换蛋白（GEP）促进ARF-GDP向ARF-GTP的转化。我们之前报道过从牛脑细胞质中分离出一种含有GEP活性的700 kDa蛋白复合物，该复合物被布雷菲德菌素A（BFA）抑制。部分纯化得到一种约60 kDa的对BFA不敏感的GEP，它增强了ARF1和ARF3与高尔基体膜的结合。现在，GEP已从大鼠脾脏细胞质中大量纯化出来，呈约55 kDa的对BFA不敏感的形式。它激活了N端肉豆蔻酰化的I类ARFs（ARFs 1和3），但不能激活来自I、II或III类的非肉豆蔻酰化的ARFs。GEP活性需要MgCl2。在存在0.6 - 0.8 mM MgCl2和1 mM EDTA的情况下，无论有无GEP，ARF1和ARF3对鸟苷5'-[γ[35S]硫代]三磷酸（[35S]GTPγS）的结合同样高。在较高的Mg2+浓度下，无GEP时的结合要低得多；在2 - 5 mM MgCl2时，GEP刺激的结合为最大值。无论有无GEP，GDP的结合速率都远低于GTPγS。磷脂对GEP活性是必需的；磷脂酰肌醇比磷脂酰丝氨酸更有效，而磷脂酸则效果较差。所测试的其他磷脂无效。最大效应需要约200 μM磷脂，在15 - 20 μM时达到半最大激活。从ARF3释放结合的[35S]GTPγS需要同时存在GEP和未标记的GTP或GTPγS；GDP的效果要差得多。Mg2+浓度和特定磷脂对纯化的对BFA不敏感的ARF GEP的显著影响的这种特性描述应有助于确定其在囊泡运输中功能的实验。
