The E3 Ubiquitin Ligases Hrd1 and gp78 Bind to and Promote Cholera Toxin Retro-Translocation
The E3 Ubiquitin Ligases Hrd1 and gp78 Bind to and Promote Cholera Toxin Retro-Translocation
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DOI：
10.1091/mbc.e09-07-0586
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发表时间：
2010-01-01
影响因子：
3.3
通讯作者：
Tsai, Billy
中科院分区：
文献类型：
--
作者：
Bernardi, Kaleena M.;Williams, Jeffrey M.;Tsai, Billy
为了引起疾病，霍乱毒素 (CT) 从细胞表面转运至内质网 (ER) 腔，其中催化 CTA1 亚基逆向易位至细胞质，诱导病理性水分泌。两个逆转录转位子成分是 Derlins 和 ER 相关的多跨 E3 泛素连接酶，包括 Hrd1 和 gp78。我们之前证明了 Derlin-1 促进 CTA1 逆转录易位。然而，由于 CTA1 在赖氨酸上及其 N 末端均未泛素化，因此 E3 连接酶在毒素逆向易位中的作用尚不清楚。在这里，我们发现突变体 Hrd1 和 gp78 以及专用于逆向易位的突变体 E2 缀合酶 (Ube2g2) 的表达会减少 CTA1 逆向易位。 Hrd1 敲低也减弱了毒素逆向易位。结合研究表明，Hrd1 和 gp78 与 CT 和蛋白质二硫键异构酶相互作用，蛋白质二硫键异构酶是一种 ER 伴侣，可展开 CTA1 以启动易位。此外，我们发现毒素与 Hrd1 和 gp78 的关联被显性阴性 Derlin-1 阻断，表明 CT 最初靶向 Derlin-1，然后转移到 Hrd1 和 gp78。这些数据证明了 E3 泛素连接酶在 CTA1 逆转录易位中的作用，暗示了内质网膜上毒素经历的一系列事件，并提出了泛素化参与转运过程的可能性。
To cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce pathological water secretion. Two retro-translocon components are the Derlins and ER-associated multi-spanning E3 ubiquitin ligases including Hrd1 and gp78. We demonstrated previously that Derlin-1 facilitates CTA1 retro-translocation. However, as CTA1 is neither ubiquitinated on lysines nor at its N-terminus, the role of E3 ligases in toxin retro-translocation is unclear. Here, we show that expression of mutant Hrd1 and gp78 and a mutant E2-conjugating enzyme dedicated to retro-translocation (Ube2g2) decrease CTA1 retro-translocation. Hrd1 knockdown also attenuated toxin retro-translocation. Binding studies demonstrate that Hrd1 and gp78 interact with CT and protein disulfide isomerase, an ER chaperone that unfolds CTA1 to initiate translocation. Moreover, we find that the toxin's association with Hrd1 and gp78 is blocked by dominant-negative Derlin-1, suggesting that CT is targeted initially to Derlin-1 and then transferred to Hrd1 and gp78. These data demonstrate a role of the E3 ubiquitin ligases in CTA1 retro-translocation, implicate a sequence of events experienced by the toxin on the ER membrane, and raise the possibility that ubiquitination is involved in the transport process.