An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii.
An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii.
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DOI：
10.1093/mp/ssn074
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发表时间：
2008-11
期刊：
影响因子：
27.5
通讯作者：
Corinne Yoshihara;Kentaro Inoue;D. Schichnes;S. Ruzin;W. Inwood;S. Kustu
中科院分区：
文献类型：
--
作者：
Corinne Yoshihara;Kentaro Inoue;D. Schichnes;S. Ruzin;W. Inwood;S. Kustu
绿色莱茵衣藻的主要Rh蛋白Rh 1与人的Rh蛋白同源。它是一种参与二氧化碳运输的膜蛋白。为了定位完整Rh 1与绿色荧光蛋白（GFP）的融合体，我们使用了一株白色（lts 1）突变的C. reinhardtii，其在类胡萝卜素生物合成的第一步被阻断。lts 1突变株积累正常量的Rh 1异养在黑暗中和Rh 1-GFP是在细胞的周边共定位与细胞质膜染料FM 4 -64。虽然Rh 1携带一个潜在的叶绿体靶向序列在其N-末端，Rh 1-GFP显然不与叶绿体包膜膜。此外，蛋白质的N-末端的一半没有进口到叶绿体在体外和N-末端地区的Rh 1没有直接进口的核酮糖二磷酸羧化酶（SSU）的小亚基。尽管我们讨论了这种解释的警告，目前的证据表明，Rh 1是一种细胞质膜蛋白，Rh 1-GFP是在C中获得的第一个细胞质膜蛋白融合。莱因哈德氏菌虽然lts 1（白色）突变株不能用于本地化的叶绿体亚区室内的蛋白质，因为他们缺乏类囊体膜，他们应该仍然是有价值的本地化许多GFP融合衣原体。
The major Rhesus (Rh) protein of the green alga Chlamydomonas reinhardtii, Rh1, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rh1 to the green fluorescent protein (GFP), we used as host a white (lts1) mutant strain of C. reinhardtii, which is blocked at the first step of carotenoid biosynthesis. The lts1 mutant strain accumulated normal amounts of Rh1 heterotrophically in the dark and Rh1-GFP was at the periphery of the cell co-localized with the cytoplasmic membrane dye FM4-64. Although Rh1 carries a potential chloroplast targeting sequence at its N-terminus, Rh1-GFP was clearly not associated with the chloroplast envelope membrane. Moreover, the N-terminal half of the protein was not imported into chloroplasts in vitro and N-terminal regions of Rh1 did not direct import of the small subunit of ribulose bisphosphate carboxylase (SSU). Despite caveats to this interpretation, which we discuss, current evidence indicates that Rh1 is a cytoplasmic membrane protein and that Rh1-GFP is among the first cytoplasmic membrane protein fusions to be obtained in C. reinhardtii. Although lts1 (white) mutant strains cannot be used to localize proteins within sub-compartments of the chloroplast because they lack thylakoid membranes, they should nonetheless be valuable for localizing many GFP fusions in Chlamydomonas.