Gile Gillian H, Keeling Patrick J
Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada.
Mol Biol Evol. 2008 Sep;25(9):1967-77. doi: 10.1093/molbev/msn147. Epub 2008 Jul 3.
Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins because none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from 7 chlorarachniophytes, and 2 distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins and which we conclude is most likely PPC targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with posttranslational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor 1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all 3 classes of targeted protein by comparing all examples of each class from expressed sequence tag surveys of B. natans and G. stellata. No recognizable difference between plastid- and PPC-targeted proteins was observed, but nucleomorph-encoded transit peptides differ, likely reflecting high AT content of nucleomorph genomes. Taken together, the data suggest that the system that directs proteins to the PPC in chlorarachniophytes uses a bipartite targeting sequence, as does the PPC-targeting system that evolved independently in cryptomonads.
绿胞藻是一类丝足虫变形鞭毛虫，它们通过捕获一种绿藻获得了光合作用能力，这种绿藻保留了一个高度简化的细胞核，称为核质体。核质体缺乏许多自身维持和表达所必需的基因，这表明一些基因已转移到宿主细胞核中，其产物现在被靶向运回周质体区室（PPC），即内共生体简化的真核细胞质。因此，绿胞藻中的蛋白质运输很复杂，包括细胞核编码的质体靶向蛋白、核质体编码的质体靶向蛋白和细胞核编码的周质体靶向蛋白。我们对这个系统理解的一个主要空白是周质体靶向蛋白，因为在任何绿胞藻中都没有相关描述。在这里，我们描述了第一个这样的蛋白，即GTP酶EFL。从7种绿胞藻中鉴定了EFL，并发现了2种不同类型。一种与有孔虫的EFL相关，且缺乏氨基末端延伸。第二种类型与之亲缘关系较远，编码一个氨基末端延伸，由一个信号肽和与细胞核编码的质体靶向蛋白的转运肽具有许多共同特征的序列组成，我们推断它最有可能靶向周质体。用针对绿胞藻“毕氏绿囊藻”和“星状裸藻”中假定的宿主和周质体靶向EFL的抗体进行蛋白质印迹分析，结果与周质体靶向蛋白前导序列的翻译后切割一致。在“毕氏绿囊藻”中对这两种蛋白进行免疫定位，证实了无前导序列的EFL位于细胞质中，而周质体靶向蛋白有独特定位模式，但不能排除其位于质体中（尽管可能性极小）。我们寻找了其他具有类似前导序列的蛋白，并鉴定了一种编码具有相同特性的二分延伸的真核翻译起始因子1。通过比较“毕氏绿囊藻”和“星状裸藻”表达序列标签调查中每一类的所有实例，对所有3类靶向蛋白的转运肽序列进行了表征。未观察到质体靶向蛋白和周质体靶向蛋白之间有可识别的差异，但核质体编码的转运肽有所不同，这可能反映了核质体基因组的高AT含量。综上所述，数据表明，绿胞藻中指导蛋白质靶向周质体的系统使用二分靶向序列，在隐藻中独立进化的周质体靶向系统也是如此。
