We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analysed with the authentic Tat substrate pOEC16, reached routinely 20-25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutated TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional analyses of protein variants. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

中文翻译：

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TatB的功能重建为类囊体Tat移位酶。

我们已经建立了用于类囊体TatB功能分析的实验系统，该系统是类囊体双精氨酸蛋白转运（Tat）机械的膜整合性TatBC受体复合物的组成部分。为此，亲和纯化的抗体抑制了分离的豌豆类囊体的固有TatB活性，并通过在试验中添加通过体外翻译获得或在大肠杆菌中异源表达后纯化的TatB蛋白来补充测定。用真实的Tat底物pOEC16分析的此类重组类囊体的Tat转运活性通常达到平行分析的模拟处理类囊体囊泡活性的20-25％。相比之下，用包含类囊体TatB的N端跨膜螺旋以外的所有抗原的纯化抗原补充测定，不会导致Tat转运重构，这证实了转运严格依赖于所添加的TatB蛋白质的活性，而不是由于内在因子的恢复通过抗体释放的TatB活性。出乎意料的是，甚至被认为无法装配到TatBC受体复合物中的突变TatB蛋白（TatB，E10C）都显示出低但可观的转运重构，这突显了该方法的敏感性及其对蛋白质变异体进一步功能分析的适用性。最后，对TatB需求的量化表明，要实现Tat依赖性类囊体转运，大约需要等摩尔量的TatA和TatB。