Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity.
Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity.
复制标题
稳定易位中间体干扰恶性疟原虫寄生虫中的全球蛋白质输出，并将 PTEX 组分 EXP2 与易位活性联系起来。
DOI：
10.1371/journal.ppat.1005618
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发表时间：
2016-05
期刊：
影响因子：
6.7
通讯作者：
Spielmann T
中科院分区：
文献类型：
--
作者：
Mesén-Ramírez P;Reinsch F;Blancke Soares A;Bergmann B;Ullrich AK;Tenzer S;Spielmann T
蛋白质输出对于细胞内恶性疟原虫血液期寄生虫的存活和毒力至关重要。为了到达宿主细胞，输出的蛋白质穿过寄生虫质膜（PPM）和寄生虫包围的寄生虫空泡膜（PVM），这是一个需要展开的过程，表明蛋白质易位。在PVM处被称为PTEX的拟议易位子的组分在该输出阶段是必不可少的，但复合物的易位活性尚未显示，并且对其拟议的膜孔组分EXP 2提出了质疑，其中在恶性疟原虫中没有功能数据。目前还不清楚PTEX如何介导可溶性蛋白和跨膜蛋白的运输。利用条件性折叠结构域，我们在这里解剖的易位事件在寄生虫的周边，显示需要两个连续的易位步骤出口的跨膜蛋白，一个在PPM和PVM之一。我们的数据提供的证据表明，根据输出的底物的C-末端的长度，这些步骤发生的PPM和PVM易位的瞬时相互作用，类似的情况下，蛋白质运输通过线粒体膜。值得注意的是，我们获得了输出蛋白的构建体，这些蛋白在跨PVM易位的过程中仍然被阻止。这堵塞了易位孔，阻止了所有类型的输出蛋白质的输出，从而抑制了寄生虫的生长。在与所提出的PTEX膜孔组分EXP 2的复合物中发现了在易位中卡住的底物，表明该蛋白在易位中的作用。这些数据首次为EXP 2是易位实体的一部分提供了证据，表明PTEX具有易位活性，并为可溶性以及跨膜蛋白从寄生虫边界转运到宿主细胞中提供了机制框架。 恶性疟原虫是人类疟疾最致命的病原体，在红细胞内发育，在红细胞内被寄生虫空泡膜（PVM）包围。为了确保细胞内存活，寄生虫将大量蛋白质输出到宿主细胞中。输出的蛋白质需要展开以穿过将寄生虫与红细胞分离的膜边界运输，这对于通过蛋白质易位膜通道运输是典型的。在这里，我们解剖了在寄生虫边界的易位事件的序列，可以有条件地阻止在易位步骤的基板。我们首次获得了在跨PVM转运过程中被捕获的输出蛋白。这就阻塞了所有其他类型的输出蛋白质的转基因座，抑制了寄生虫的生长。在易位中卡住的构建体与EXP 2形成复合物，EXP 2是已知对蛋白质输出必不可少的复合物的组分，称为PTEX。我们的工作链接的需要展开和这个复杂的功能在出口，实验证据表明，PTEX确实是一个translocon。连接的不可折叠结构域在解开跨膜运输过程中起着重要作用，并在此解决了不同种类的输出蛋白到达恶性疟原虫感染的宿主细胞所需的运输步骤。
Protein export is central for the survival and virulence of intracellular P. falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export but translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Our data provide evidence that, depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data for the first time provide evidence for EXP2 to be part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell. P. falciparum parasites, the deadliest agent of human malaria, develop within erythrocytes where they are surrounded by a parasitophorous vacuolar membrane (PVM). To ensure intracellular survival, the parasite exports a large repertoire of proteins into the host cell. Exported proteins require unfolding for trafficking across the membrane boundaries separating the parasite from the erythrocyte, typical for transport by protein translocating membrane channels. Here, we dissected the sequence of translocation events at the parasite boundary using substrates that can be conditionally arrested at translocation steps. We for the first time obtained exported proteins arrested in the process of being translocated across the PVM. This jammed the translocons for all other types of exported proteins and inhibited parasite growth. The constructs stuck in translocation were in a complex with EXP2, a component of a complex known to be essential for protein export that is termed PTEX. Our work links the need for unfolding and the function of this complex in export, giving experimental evidence that PTEX indeed is a translocon. Conditionally unfoldable domains have been instrumental in unravelling transport processes across membranes and here resolve the transport steps the different kinds of exported proteins require to reach the P. falciparum-infected host cell.