ID OF CYTOSOLIC TRANSLOCATION FACTORS INVOLVED IN BACTERIAL INTOXIFICATION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。白喉毒素催化（C）结构域从纯化的早期内体的内腔到外部环境的体外递送需要添加ATP和胞质易位因子（CTF）复合物。使用跨内体膜的C-结构域ADP-核糖基转移酶活性的易位作为测定，CTF复合物活性分别从人T细胞和酵母提取物纯化650-800倍。用MALDI、ESI和毛细管LC/MS鉴定了从人T细胞和酵母中纯化的CTF复合物中的伴侣蛋白热休克蛋白（Hsp）90和硫氧还蛋白还原酶。在体外易位试验和完整细胞毒性试验中，对这两种蛋白质与特异性抑制剂所起的作用的进一步分析已经证明了它们在C结构域从早期内体的内腔到外部环境的生产性递送中的重要作用。这些结果证实并扩展了白喉毒素在C-结构域展开和重折叠，必须发生之前和之后囊泡膜易位早期观察。此外，这里提出的结果表明，硫氧还蛋白还原酶活性在C-结构域的胞质释放中起着至关重要的作用。由于类似的CTF复合物已部分纯化的哺乳动物和酵母细胞提取物，这里提出的结果表明一个共同的和基本的机制，跨早期内体膜的C-结构域易位。这些结果已发表在《细胞生物学杂志》上。对CTF的剩余蛋白质成分的持续分析已经产生了关于参与细菌增强机制的其他成分的信息。在这项研究中发现的机制正在转向我们在其他系统中的普遍应用，JCB论文已被引用超过21次。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by MALDI, ESI, and capillary LC/MS in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria tox in C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes. These results have been published in J. Cell Biol. Continued analysis of the remaining protein constituents of the CTFs has yielded information on additional components that are involved in the mechanisms of bacterial intoxification. The mechansms uncovered in this study are turning our have general applications in other systems and more than 21 citations have been made to the JCB paper.