Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system
Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system
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DOI：
10.1083/jcb.201812020
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发表时间：
2019-03-01
影响因子：
7.8
通讯作者：
De Matteis, Maria Antonietta
中科院分区：
文献类型：
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作者：
Venditti, Rossella;Rega, Laura Rita;De Matteis, Maria Antonietta
Er-TGN接触点(ERTGoCs)已被电子显微镜观察到，但它们位于密集的核周区域，阻碍了它们的光学显微镜分析和机制研究。为了克服这些限制，我们开发了一种基于FRET的方法，并筛选了几个候选基因来搜索ERTGoCS的分子决定因素。这些包括ER膜蛋白VAPA和VAPB，以及具有双重靶向基序的脂转移蛋白(ER和TGN)，这些蛋白被认为有助于维持ERTGoCS，如神经酰胺转移蛋白CERT和几个氧固醇结合蛋白成员。我们发现VAP蛋白OSBP1、ORP9和ORP10是必需的，其中OSBP1与ORP9起冗余作用，ORP9不参与其脂质转移活性，而ORP10由于其将磷脂酰丝氨酸转移到TGN的能力而被需要。我们的结果表明，ERTGoCS的完整性既需要结构锚链，也需要适当的脂质成分。
ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.