Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea
Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea
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DOI：
10.1128/jb.187.23.8104-8113.2005
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发表时间：
2005-12-01
影响因子：
3.2
通讯作者：
Pohlschröder, M
中科院分区：
文献类型：
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作者：
Dilks, M;Giménez, MI;Pohlschröder, M
双精氨酸转位(TAT)途径广泛存在于多种原核生物中，能够从细胞质中输出部分或全部折叠的蛋白质。尽管不同类别的蛋白质通过TAT途径运输，但在大多数生物体中，与SEC途径相比，TAT途径促进了相对较少数量的底物的分泌。然而，计算证据表明，嗜盐古菌将几乎所有分泌的蛋白质都路由到TAT途径。我们扩展了以前对嗜盐古生菌TAT途径的计算分析，并开始在体内表征模型卤虫考古菌的TAT机制，即Haloferax Volcann II。与这一途径在盐生古生菌中的预测用途一致，我们确定了在盐生真菌中发现的四个TAT基因中的三个对于在复杂介质中有氧生长时的生存是必不可少的。这是首次报道一种在这种条件下生长时需要TAT途径才能存活的有机体。非必需基因的缺失对TAT途径已证实的底物的分泌没有影响。两个TATA类似物TatAo和Tatat在细胞膜和细胞质中均可检测到，并可从细胞质中分离出来。利用尺寸排斥层析进一步鉴定细胞质和细胞膜TATA蛋白，我们发现这些蛋白存在于这两个细胞组分的高分子量复合体中。
The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.