Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells
Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells
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DOI：
10.1128/jvi.76.20.10485-10496.2002
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发表时间：
2002-10
影响因子：
5.4
通讯作者：
F. Weber-Lotfi;A. Dietrich;M. Russo;L. Rubino
中科院分区：
文献类型：
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作者：
F. Weber-Lotfi;A. Dietrich;M. Russo;L. Rubino
康乃馨意大利环斑病毒基因组RNA在植物细胞中的复制发生在多泡体中，多泡体在感染过程中从线粒体外膜发育而来。病毒基因组中的ORF 1编码36-kDa蛋白，而ORF 2通过ORF 1终止密码子的通读编码95-kDa复制酶。我们以前已经表明，ORF 1的N-末端部分包含导致线粒体囊泡化的信息，并且36-kDa蛋白定位于线粒体。使用感染，在植物和酵母细胞中的绿色荧光蛋白融合体的体内表达，并在体外线粒体整合测定，我们在这里证明，这两个36-kDa的蛋白质和完整的复制酶都有针对性的线粒体和锚的N端和C端的细胞溶质侧的外膜。缺失突变体的分析表明，锚序列可能大致对应于氨基酸84至196，含有两个跨膜结构域。没有发现基质靶向前序列的证据，数据表明，病毒蛋白的膜插入是由依赖于ORF 1的N-末端部分中存在的两个跨膜片段和多个识别信号的输入受体无关的信号锚定机制介导的。
ABSTRACT Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.