使用重组酵母膜的多胺转运测定
Polyamine Transport Assay Using Reconstituted Yeast Membranes.
作者信息
Van Veen Sarah, Martin Shaun, Schuermans Marleen, Vangheluwe Peter
机构信息
Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.
出版信息
Bio Protoc. 2021 Jan 20;11(2):e3888. doi: 10.21769/BioProtoc.3888.
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 , 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
ATP13A2/PARK9是一种晚期内体/溶酶体P5B转运ATP酶，与多种神经退行性疾病相关。我们最近将ATP13A2鉴定为溶酶体多胺输出蛋白，这为未知的哺乳动物多胺转运系统的分子特性提供了线索。在这里，我们逐步描述了一种用于测量过表达人ATP13A2的酵母细胞重构囊泡中放射性标记多胺转运的实验方案。该方案是我们最近发表的论文（van Veen，2020）的一部分，对于表征其他假定的多胺转运蛋白（如P5B转运ATP酶的同工型）的转运功能将是有用的。
相似文献
1
Polyamine Transport Assay Using Reconstituted Yeast Membranes.使用重组酵母膜的多胺转运测定
Bio Protoc. 2021 Jan 20;11(2):e3888. doi: 10.21769/BioProtoc.3888.
2
ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress.ATP13A2 介导的内溶酶体多胺输出可抵抗线粒体氧化应激。
Proc Natl Acad Sci U S A. 2020 Dec 8;117(49):31198-31207. doi: 10.1073/pnas.1922342117. Epub 2020 Nov 23.
3
Structural basis of polyamine transport by human ATP13A2 (PARK9).人源 ATP13A2（PARK9）转运多胺的结构基础。
Mol Cell. 2021 Nov 18;81(22):4635-4649.e8. doi: 10.1016/j.molcel.2021.08.017. Epub 2021 Oct 28.
4
Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2.人类P5B型ATP酶ATP13A2的冷冻电镜结构及转运机制
Cell Discov. 2021 Nov 2;7(1):106. doi: 10.1038/s41421-021-00334-6.
5
ATP13A2 deficiency disrupts lysosomal polyamine export.ATP13A2 缺陷破坏溶酶体多胺输出。
Nature. 2020 Feb;578(7795):419-424. doi: 10.1038/s41586-020-1968-7. Epub 2020 Jan 29.
6
Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2.低温电子显微镜揭示了人类 ATP13A2 通过脂筏促进多胺输出的机制见解。
Mol Cell. 2021 Dec 2;81(23):4799-4809.e5. doi: 10.1016/j.molcel.2021.11.001. Epub 2021 Nov 18.
7
Structural mechanisms for gating and ion selectivity of the human polyamine transporter ATP13A2.人多胺转运体 ATP13A2 的门控和离子选择性的结构机制。
Mol Cell. 2021 Nov 18;81(22):4650-4662.e4. doi: 10.1016/j.molcel.2021.10.002. Epub 2021 Oct 28.
8
Parkinson disease related ATP13A2 evolved early in animal evolution.帕金森病相关 ATP13A2 在动物进化早期就已经出现。
PLoS One. 2018 Mar 5;13(3):e0193228. doi: 10.1371/journal.pone.0193228. eCollection 2018.
9
Parkinson's disease-associated human P5B-ATPase ATP13A2 increases spermidine uptake.帕金森病相关的人源 P5B-ATP 酶 ATP13A2 增加精脒摄取。
Biochem J. 2013 Feb 15;450(1):47-53. doi: 10.1042/BJ20120739.
10
Structure and transport mechanism of P5B-ATPases.P5B-ATPases 的结构与转运机制。
Nat Commun. 2021 Jun 25;12(1):3973. doi: 10.1038/s41467-021-24148-y.
引用本文的文献
1
A P5-ATPase, TgFLP12, diverging from plant chloroplast lipid transporters mediates apicoplast fatty export in Toxoplasma.一种与植物叶绿体脂质转运蛋白不同的P5-ATP酶TgFLP12介导了弓形虫顶质体的脂肪酸输出。
Nat Commun. 2025 Jul 1;16(1):5538. doi: 10.1038/s41467-025-61155-9.
2
ATP13A2 modifies mitochondrial localization of overexpressed TOM20 to autolysosomal pathway.ATP13A2 将过表达的 TOM20 修饰到自噬溶酶体途径中的线粒体定位。
PLoS One. 2022 Nov 29;17(11):e0276823. doi: 10.1371/journal.pone.0276823. eCollection 2022.
本文引用的文献
1
ATP13A2 deficiency disrupts lysosomal polyamine export.ATP13A2 缺陷破坏溶酶体多胺输出。
Nature. 2020 Feb;578(7795):419-424. doi: 10.1038/s41586-020-1968-7. Epub 2020 Jan 29.
2
A lipid switch unlocks Parkinson's disease-associated ATP13A2.一种脂质开关激活与帕金森病相关的ATP13A2。
Proc Natl Acad Sci U S A. 2015 Jul 21;112(29):9040-5. doi: 10.1073/pnas.1508220112. Epub 2015 Jul 1.
3
A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.一种用于纯化完整的Drs2p-Cdc50p脂质翻转酶复合物的高效共表达系统，该系统严重依赖磷脂酰肌醇-4-磷酸并由其稳定。
PLoS One. 2014 Nov 13;9(11):e112176. doi: 10.1371/journal.pone.0112176. eCollection 2014.
4
Measuring Ca2+-dependent Ca2+-uptake activity in the mouse heart.测量小鼠心脏中钙离子依赖性钙离子摄取活性。
Cold Spring Harb Protoc. 2014 Aug 1;2014(8):876-86. doi: 10.1101/pdb.prot076893.
5
Pathogenic effects of novel mutations in the P-type ATPase ATP13A2 (PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism.导致 Kufor-Rakeb 综合征（一种早发性帕金森病）的 P 型 ATP 酶 ATP13A2（PARK9）新型突变的致病作用。
Hum Mutat. 2011 Aug;32(8):956-64. doi: 10.1002/humu.21527. Epub 2011 Jul 12.
6
Polyamine transport systems in mammalian cells and tissues.哺乳动物细胞和组织中的多胺转运系统。
Methods Mol Biol. 2011;720:339-48. doi: 10.1007/978-1-61779-034-8_21.
7
Novel ATP13A2 variant associated with Parkinson disease in Taiwan and Singapore.在台湾和新加坡发现的与帕金森病相关的新型ATP13A2变体。
Neurology. 2008 Nov 18;71(21):1727-32. doi: 10.1212/01.wnl.0000335167.72412.68.
8
ATP13A2 missense mutations in juvenile parkinsonism and young onset Parkinson disease.青少年帕金森病和早发性帕金森病中的ATP13A2错义突变。
Neurology. 2007 May 8;68(19):1557-62. doi: 10.1212/01.wnl.0000260963.08711.08.
9
Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles.通过巨型单层囊泡的形状变化，用未标记的脂质检测翻转酶活性。
J Biol Chem. 2007 May 25;282(21):15559-68. doi: 10.1074/jbc.M604740200. Epub 2007 Mar 17.
10
Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase.伴有痴呆的遗传性帕金森病由编码溶酶体5型P型ATP酶的ATP13A2基因突变引起。
Nat Genet. 2006 Oct;38(10):1184-91. doi: 10.1038/ng1884. Epub 2006 Sep 10.
Zotero 插件