Polyamine Transport Assay Using Reconstituted Yeast Membranes
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Author:
Date:
2021-01-20
[Abstract] ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.
[摘要] [摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase,与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者,这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里,我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分(van Veen等,2020),将有助于表征其他假定的多胺转运蛋白的转运功能,例如P5B转运ATPase的同工型。
[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白,与一系列神经退行性疾病有关,例如早发性帕金森氏病(Di Fonzo等,2007 ;Lin等,2008)和Kufor -Rakeb综合征(伴痴呆的早期帕金森病)(Ramirez等,2006 ;Park等,2011)。ATP13A2属于P型转运ATPase ,是一类活性转运蛋白,由于ATP水解而暂时形成磷酸中间产物(Kuhlbrandt ,2004年)。ATP13A2是P5亚家族的成员,该家族已在20多年前通过基因组测序鉴定出来(Axelsen和Palmgren ...
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Purification of Rice Stripe Virus
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Author:
Date:
2020-03-20
[Abstract] Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated with polyethylene glycol (PEG) followed by pelleting through primary ultracentrifugation, ultracentrifugation in a glycerol cushion and ultracentrifugation in density gradient. The purified RSV virions can not only be viewed as filamentous particles under an electron microscope, but can also be acquired by insect vector through direct injection into insect body or through membrane feeding prior to transmission to rice plants.
[摘要] [摘要 ] 尽管现在可以使用许多球形和杆状植物病毒的纯化方案,但有关丝状植物病毒纯化的协议却只有少数。在这里,我们提出一个协议水稻的大规模纯化小号牛肚v IRUS 从RSV感染的水稻组织(RSV)。首先用聚乙二醇(PEG)沉淀具有高感染力的RSV 病毒颗粒,然后通过一次超速离心,在甘油垫层中超速离心和密度梯度超速离心沉淀。纯化的RSV 病毒体不仅可以在电子显微镜下观察为丝状颗粒,而且还可以通过昆虫载体直接注入昆虫体内或在送入水稻之前通过膜饲喂获得。
[背景 ] 许多purificatio n,用于球形和棒状病毒协议已被发表(安德烈é 等人,2002; BALKE 。等人,2018) 。这些协议都依赖于化学沉淀或密度梯度离心。但是,丝状病毒的纯化方案目前受到限制。
大米小号肚v 病毒属(RSV)是负链RNA病毒,属于属纤细病毒,订单Bunyavirales 。在许多东亚国家,RSV经常对水稻生产造成严重损害(Whitfield 等,2015; Liu 等,2018)。与生产球状和包膜病毒体的Bunyavirales 顺序中的其他成员不同,RSV 病毒体是丝状的。然而,RSV基因组编码在纯化的RSV 病毒体中未发现的糖蛋白(Toriyama,19 86; Lu 等人,2019)。已知RSV ...
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