Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

作     者：Hongmei Ke Zhi Feng Min Liu Tianhui Sun Jianli Dai Mengqi Ma Lu-Ping Liu Jian-Quan Ni Jos Carlos Pastor-Pareja 

作者机构：School of Life SciencesTsinghua University School of MedicineTsinghua University 

出 版 物：《Journal of Genetics and Genomics》 (遗传学报（英文版）)

年 卷 期：2018年第45卷第6期

页      面：299-313页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 

基　　金：funded by grants from the Natural Science Foundation of China (Nos.31771600 and 31750410689) Tsinghua Initiative Program (No.20131089281) a 1000 Talents award,all to J.C.P.-P 

主　　题：Collagen Ⅳ COPⅡ vesicles RNAi screening Secretory pathway Rab-GTPases ER exit site 

摘      要：Collagens are large secreted trimeric proteins making up most of the animal extracellular *** 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 ***, 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 a 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 Rab X3, 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.