Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Mol Biol Cell. 2010 Apr 1;21(7):1305-13. doi: 10.1091/mbc.e09-09-0826. Epub 2010 Feb 3.
Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. Our previous analyses demonstrated that the ER oxidoreductase protein disulfide isomerase (PDI) acts as a redox-dependent chaperone to unfold CTA1, a reaction postulated to initiate toxin retro-translocation. In its reduced state, PDI binds and unfolds CTA1; subsequent oxidation of PDI by Ero1alpha enables toxin release. Whether this in vitro model describes events in cells that control CTA1 retro-translocation is unknown. Here we show that down-regulation of Ero1alpha decreases retro-translocation of CTA1 by increasing reduced PDI and blocking efficient toxin release. Overexpression of Ero1alpha also attenuates CTA1 retro-translocation, an effect due to increased PDI oxidation, which prevents PDI from engaging the toxin effectively. Interestingly, Ero1alpha down-regulation increases interaction between PDI and Derlin-1, an ER membrane protein that is a component of the retro-translocation complex. These findings demonstrate that an appropriate Ero1alpha-PDI ratio is critical for regulating the binding-release cycle of CTA1 by PDI during retro-translocation, and implicate PDI's redox state in targeting it to the retro-translocon.
霍乱毒素（CT）从宿主细胞的质膜运输到内质网（ER），在那里催化 CTA1 亚基逆行转运到细胞质以诱导毒性。我们之前的分析表明，内质网氧化还原酶蛋白二硫键异构酶（PDI）作为一种依赖于氧化还原的伴侣，可展开 CTA1，这一反应被假设为毒素逆行转运的起始。在其还原状态下，PDI 结合并展开 CTA1；随后 Ero1alpha 对 PDI 的氧化使毒素释放。这种体外模型是否描述了控制 CTA1 逆行转运的细胞内事件尚不清楚。在这里，我们表明 Ero1alpha 的下调通过增加还原型 PDI 和阻止有效毒素释放来减少 CTA1 的逆行转运。Ero1alpha 的过表达也会减弱 CTA1 的逆行转运，这是由于 PDI 氧化增加所致，这阻止了 PDI 与 Derlin-1 有效地结合，Derlin-1 是内质网膜蛋白，是逆行转运复合物的组成部分。这些发现表明，在逆行转运过程中，适当的 Ero1alpha-PDI 比值对于调节 PDI 与 CTA1 的结合-释放循环至关重要，并暗示 PDI 的氧化还原状态将其靶向逆行转运体。
