ClpA mediates directional translocation of substrate proteins into the ClpP protease
ClpA mediates directional translocation of substrate proteins into the ClpP protease
复制标题
ClpA 介导底物蛋白定向易位至 ClpP 蛋白酶中
DOI：
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发表时间：
2001
影响因子：
11.1
通讯作者：
E. Weber
中科院分区：
文献类型：
--
作者：
B. Reid;W. Fenton;A. Horwich;E. Weber
许多蛋白质的细胞内降解以 ATP 依赖性方式通过包含与蛋白水解圆柱体同轴相关的分子伴侣环复合物的大组件介导，例如原核生物中的 ClpAP、ClpXP 和 HslUV，以及真核生物中的 26S 蛋白酶体。最近对分子伴侣 ClpA 的研究表明，它介导底物蛋白的 ATP 依赖性解折叠，并指导其 ATP 依赖性易位至 ClpP 蛋白酶中。由于进入蛋白水解室的轴向通道很窄，因此未折叠的底物蛋白似乎有可能从分子伴侣穿入蛋白酶中，这表明易位可能是定向的。我们通过使用带有 COOH 末端 ssrA 识别元件的两种底物蛋白研究了 ClpA/ClpP 介导的反应的方向性，每个底物蛋白都用荧光探针标记在 NH2 或 COOH 末端附近。测量 ClpP 腔中供体荧光团与作为受体的底物探针之间荧光各向异性和荧光共振能量转移的时间依赖性变化，以监测底物从 ClpA 到 ClpP 的易位。我们观察到，对于两种底物，COOH 末端标记的分子比 NH2 末端标记的分子能量转移发生的时间要早 2-4 秒，这表明易位确实是定向的，底物蛋白的 COOH 末端首先进入 ClpP。
The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH2 or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2–4 s sooner with the COOH-terminally labeled molecules than with the NH2-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.
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