Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon
Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon
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DOI：
10.1128/jb.00493-18
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发表时间：
2019-01-01
影响因子：
3.2
通讯作者：
Randall, Linda L.
中科院分区：
文献类型：
--
作者：
Bariya, Priya;Randall, Linda L.
在所有细胞中，高度保守的通道跨膜运输蛋白质。在大肠杆菌中，该通道是 SecYEG。许多对该蛋白质复合物的研究都使用了重组为蛋白脂质体的纯化 SecYEG。重构系统的活性如何准确地反映原生膜环境中 SecYEG 的特性？我们通过比较三种体外系统进行研究：内膜囊泡的天然膜环境和两种重建方法。一种广泛使用的方法是单独将 SecYEG 重构为脂质双层。另一种是我们将 SecYEG 与 SecA（转位酶的 ATP 酶）共组装的方法。对于九种不同的前体物种，我们评估了表征易位的参数：有能力的前体易位的最大幅度、能量与转移的耦合以及表观速率常数。此外，我们还研究了伴侣 SecB 存在和不存在时的易位。对于所有九种前体，对于所研究的每个参数，与 SecA 共组装的 SecYEG 与天然膜中的 SecYEG 一样活跃。 SecB 对前体转运的影响忠实地模拟了体内观察结果。通过对九种不同前体的研究，我们得出结论，表观速率常数反映了限制易位速率的步骤，取决于与前导体以外的前体部分易位子的相互作用。此外，在某些情况下，限速步骤会因 SecB 的存在而改变。讨论了与我们的数据一致的限速步骤的候选者。重要性这项工作对三个体外系统中的 Sec 分泌系统的运输参数进行了全面的量化。只需在重构过程中将 SecA 添加到 SecYEG 中，大多数研究人员使用的标准重构即可增强，产生六倍的活性易位子。这个强大的系统忠实地反映了天然膜囊泡的易位特性。我们已将研究的前体数量扩大到九个。这使我们得出结论，易位的速率常数随前体物种的不同而变化。
In all cells, a highly conserved channel transports proteins across membranes. In Escherichia coil, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment ? We investigated by comparing three in vitro systems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations made in vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCE This work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the three in vitro systems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.