ID OF CYTOSOLIC TRANSLOCATION FACTORS INVOLVED IN BACTERIAL INTOXIFICATION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在体外将白喉毒素催化(C)结构域从纯化的早期内体的管腔传递到外部环境需要ATP和细胞质易位因子（CTF）复合物的加入。利用c结构域adp -核糖基转移酶活性跨内体膜的易位作为测定，从人T细胞和酵母提取物中分别纯化出650-800倍的CTF复合物活性。采用MALDI、ESI和毛细管LC/MS对从人T细胞和酵母中纯化的CTF复合物进行了伴蛋白热休克蛋白（Hsp） 90和硫氧还蛋白还原酶的鉴定。在体外易位试验和完整细胞毒性试验中，对这两种蛋白与特异性抑制剂的进一步分析表明，它们在c结构域从早期内体的管腔到外部环境的有效传递中发挥了重要作用。这些结果证实并扩展了白喉毒素在囊泡膜易位之前和之后必须发生的c结构域展开和重折叠的早期观察。此外，本文的结果表明，硫氧还蛋白还原酶活性在c结构域的胞质释放中起着重要作用。由于类似的CTF复合物已经从哺乳动物和酵母细胞提取物中部分纯化，因此本文的研究结果表明，c结构域在早期内体膜上易位的共同和基本机制。这些研究结果发表在《细胞生物学》杂志上。对CTFs剩余蛋白质成分的持续分析已经获得了参与细菌中毒机制的其他成分的信息。本研究中发现的机制正在其他系统中得到普遍应用，JCB论文被引用超过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.