Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements
Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements
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果蝇中的胶原蛋白分泌筛选支持共同的分泌机制和多种 Rab 需求
DOI：
10.1016/j.jgg.2018.05.002
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发表时间：
2018-06-20
影响因子：
5.9
通讯作者：
Pastor-Pareja, Jose Carlos
中科院分区：
文献类型：
--
作者：
Ke, Hongmei;Feng, Zhi;Pastor-Pareja, Jose Carlos
胶原蛋白是构成动物细胞外基质大部分的大型分泌三聚体蛋白。近年来，胶原蛋白的分泌一直是细胞生物学家关注的焦点，因为胶原蛋白三聚体太大且坚硬，无法装入介导从内质网 (ER) 到高尔基体运输的 COPII 囊泡中。已经假设了创建扩大的 ER 至高尔基体运输载体的胶原蛋白特异性机制，包括通过保守的 ER 出口位点 (ERES) 蛋白 Tango1 进行货物装载。在这里，我们报告了对果蝇胶原蛋白分泌相关基因的 RNAi 筛选。在这次筛选中，我们检查了 GFP 标记的 IV 型胶原蛋白在活体动物中的分布，发现了 88 个基因命中，这些基因的敲低导致 IV 型胶原蛋白在脂肪体内的细胞内积累，而脂肪体是幼虫基质蛋白的主要来源。在这些命中中，只有两个特别影响胶原蛋白的分泌：PH4 α EFB 和 Plod，编码已知可介导 ER 中胶原蛋白翻译后修饰的酶。每一次细胞内积累都会影响一般分泌，这与胶原蛋白的分泌不使用特定的囊泡运输模式，而是使用一般分泌途径的概念一致。我们的热门产品包括真核分泌机制中的许多已知参与者，例如 COPII 和 COPI 组件、SNARE 和 Rab-GTPase 调节器。我们对 Rab-GTP 酶参与分泌的进一步分析表明，Rab1、Rab2 和 RabX3 都是 ERES 所必需的，它们各自对 ERES 形态有不同的影响。相比之下，通过 Rep 敲低来消除这三种活性会导致 ERES 和高尔基体解偶联。我们还介绍了我们命名为 trabuco (tbc) 的筛选命中的特征，它编码包含 Rab-GAP 的 ERES 本地化 TBC 结构域。最后，与之前在果蝇 S2 细胞中进行的两次分泌筛选相比，我们讨论了我们的筛选在识别分泌途径基因方面的成功。版权所有(C) 2018，中国科学院遗传与发育生物学研究所，中国遗传学会。由爱思唯尔有限公司和科学出版社出版。版权所有。
Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4 alpha EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells. Copyright (C) 2018, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.