Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.

Arf蛋白是小的Ras家族GTPases，可将网格蛋白和COPI外壳吸收到高尔基体膜上，并调节膜运输机制的组成部分。据信，Alfs的膜缔合和活性与GTP结合偶联，其中GTP水解为囊泡脱膜所需。在人类中，四种Arf蛋白（Arf1，Arf3，Arf4和Arf5）与高尔基体相关。相互矛盾的报告表明，与布雷菲德菌素A敏感的交换因子GBF1无关，HA-GFP标记的II类ARF（Arf4和Arf5）被募集到膜上，这提示调节与I类Arfs（Arf1，Arf3）存在根本性的差异，或者GFP标记的II类Arfs的GTPase循环与其他Arfs相似。我们证明了这些结果取决于荧光标记，而Arf4-HA-GFP标记对布雷菲德菌具有抗性，但Arf4-GFP在布雷菲德灵敏性和光漂白试验中的作用类似于Arf1-GFP。通过将HA标签中的两个天冬氨酸突变为丙氨酸，可以将Arf4-HA-GFP部分还原为Arf4-GFP的行为。我们的结果表明Arf4对标签具有很高的敏感性，可以解释先前研究之间的差异。我们讨论了这项研究对标记Arfs未来工作的意义。