Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex.
Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex.
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DOI：
10.1186/1471-2121-9-54
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发表时间：
2008-09-23
期刊：
影响因子：
--
通讯作者：
Mullen RT
中科院分区：
文献类型：
--
作者：
Hwang YT;McCartney AW;Gidda SK;Mullen RT
香石竹意大利环斑病毒（CIRV）是一种正链RNA病毒，其在感染的宿主细胞中引起线粒体的大规模结构改变，最显著的是形成许多内部囊泡/小球，这些囊泡/小球来源于线粒体外膜并作为病毒RNA复制的位点。虽然CIRV复制复合物的膜结合组分，包括36-kD RNA结合蛋白（p36），已知对线粒体形态的这些变化是必不可少的，并且在新生病毒RNA合成中的作用方面相对较好地表征，但这些蛋白质如何特异性靶向并插入线粒体的定义很差。在这里，我们报告的分子信号负责分选p36的线粒体外膜。使用功能获得性测定与部分p36融合的报告蛋白和结构域交换测定与p36和另一个密切相关的病毒RNA结合蛋白，p33，特别是过氧化物酶体边界膜的组合，我们表明，在p36的线粒体靶向信息驻留在其两个跨膜结构域（TMD）和插入亲水环序列。对p36中这些区域的综合突变分析表明，主要靶向决定因素是两种TMD的中等疏水性和间插环序列内两亲性螺旋的带正电荷的面。我们还使用双分子荧光互补（BiFC）表明，p36与线粒体外膜（TOM）中移位酶复合物的某些组分相互作用，但不与分选和组装机制（SAM）相互作用。我们的研究结果提供了深入了解病毒，如CIRV，如何利用特定的宿主细胞蛋白分选途径，以促进其复制。p36靶向和插入线粒体外膜的表征也揭示了将宿主细胞膜蛋白分选到线粒体中所涉及的机制，这是一个在植物中基本上未被探索的过程。
Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM). Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.
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DOI：
10.1073/pnas.0506958103
发表时间：
2006-04-25
影响因子：
11.1
作者：
Dunkley, TPJ;Hester, S;Lilley, KS
通讯作者：
Lilley, KS
影响因子：
3.8
作者：
Burgyan, J;Rubino, L;Russo, M
通讯作者：
Russo, M
DOI：
10.1016/s0889-1605(86)80004-0
发表时间：
1986-10-01
期刊：
JOURNAL OF ULTRASTRUCTURE AND MOLECULAR STRUCTURE RESEARCH
影响因子：
--
作者：
APPIANO, A;DAGOSTINO, G;DELLORTO, P
通讯作者：
DELLORTO, P
DOI：
10.1007/978-1-59745-365-3_38
发表时间：
2007-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Heazlewood, Joshua L;Millar, A Harvey
通讯作者：
Millar, A Harvey
影响因子：
11.6
作者：
Heazlewood, JL;Tonti-Filippini, JS;Millar, AH
通讯作者：
Millar, AH