Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells:: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1
Characterization of class I and II ADP-ribosylation factors (Arfs) in live cells:: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1
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DOI：
10.1091/mbc.e08-04-0373
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发表时间：
2008-08-01
影响因子：
3.3
通讯作者：
Melancon, Paul
中科院分区：
文献类型：
--
作者：
Chun, Justin;Shapovalova, Zoya;Melancon, Paul
尽管对高尔基复合体中的 ADP-核糖基化因子 (Arf) 1 进行了大量研究，但 Arf2-5 在分泌途径中的功能或任何 Arf 在 ER-高尔基中间室 (ERGIC) 中的功能仍然未知。在这里，我们检查了荧光标记的 Arf1、-3、-4 和 -5 招募到外周 ERGIC 上的情况。活细胞成像检测到外周泪点上的 Arfs，其中还含有高尔基体特异性布雷菲德菌素 A (BFA) 抗性因子 (GBF) 1 和 ERGIC 标记 p58。出乎意料的是，BFA 并没有促进 Arfs 与 GBF1 在高尔基复合体或 ERGIC 的共招募，但它发现了 Arf1,3 和 Arf4,5 之间的显着差异。尽管添加 BFA 后 Arf1,3 很快与所有内膜解离，但 Arf4,5 仍保留在 ERGIC 结构上，即使在 GBF1 重新分配到单独的区室后也是如此。即使存在 BFA 和 Exo1，GDP 抑制的 Arf4(T31N) 突变体仍定位于 ERGIC。此外，用 Exo1 处理后 Arf 中心点 GTP 的丢失导致所有 Arf 从高尔基复合体中快速释放，并导致 GBF1 在高尔基体和 ERGIC 膜上积累。我们的结果表明，GDP 结合的 Arf4,5 通过与负责 GBF1 募集的结合位点不同的结合位点与 ERGIC 膜结合。此外，他们提供了第一个证据表明GBF1在膜上的积累可能是由于Arf中心点GTP的丢失引起的，而不是Arf中心点GDP中心点BFA中心点GBF1复合物的形成造成的。
Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2-5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf center dot GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf center dot GTP, rather than the formation of an Arf center dot GDP center dot BFA center dot GBF1 complex.