PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila
PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila
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DOI：
10.1371/journal.pone.0142562
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发表时间：
2015-11-10
期刊：
影响因子：
3.7
通讯作者：
Harris, Tony J. C.
中科院分区：
文献类型：
--
作者：
Lee, Donghoon M.;Rodrigues, Francisco F.;Harris, Tony J. C.
GDP/GTP交换因子（GEFs）募集到特定的亚细胞位点决定了它们在哪里激活小G蛋白来调节各种细胞过程。细胞分裂素是一个保守的质膜gef家族，用于调节内吞作用的Arf小G蛋白。对哺乳动物细胞分裂素的分析已经确定了这些多结构域蛋白的一些募集机制，但这些机制的保护和发育作用尚不清楚。在这里，我们报告了果蝇细胞分裂素Steppke的pleckstrin同源（PH）结构域如何影响其在早期胚胎卵裂沟的定位和活性。我们发现PH结构域是Steppke沟定位和调节沟结构所必需的。然而，PH结构域不足以进行定位。接下来，我们研究了哺乳动物细胞分裂素结合PIP3或gtp结合的Arf - G蛋白所需的保守PH结构域氨基酸残基的作用。我们证实Steppke PH结构域在体外通过保守机制优先结合PIP3。然而，破坏PIP3结合残基对GFP-Steppke的定位和作用没有明显影响。相反，与gtp结合的arfg蛋白结合的残基对这种Steppke定位和活性做出了主要贡献。通过分析gfp标记的Arf和Arf样小G蛋白，我们发现Arf1-GFP、Arf6-GFP和Arl4-GFP定位在犁沟上，但Arf4-GFP不定位。然而，对Arf1， Arf6或ar14缺失的胚胎的分析显示，要么比Steppke缺失的胚胎更早出现缺陷，要么没有可检测到的沟缺陷，可能是因为冗余，因此很难评估单个Arf小G蛋白如何影响Steppke。尽管如此，我们的数据表明，Steppke PH结构域及其与gtp结合的Arf G蛋白结合的保守残基对早期果蝇胚胎中Steppke的定位和活性有实质性影响。
The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.