Bernardi Kaleena M, Forster Michele L, Lencer Wayne I, Tsai Billy
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Mol Biol Cell. 2008 Mar;19(3):877-84. doi: 10.1091/mbc.e07-08-0755. Epub 2007 Dec 19.
Cholera toxin (CT) intoxicates cells by using its receptor-binding B subunit (CTB) to traffic from the plasma membrane to the endoplasmic reticulum (ER). In this compartment, the catalytic A1 subunit (CTA1) is unfolded by protein disulfide isomerase (PDI) and retro-translocated to the cytosol where it triggers a signaling cascade, leading to secretory diarrhea. How CT is targeted to the site of retro-translocation in the ER membrane to initiate translocation is unclear. Using a semipermeabilized-cell retro-translocation assay, we demonstrate that a dominant-negative Derlin-1-YFP fusion protein attenuates the ER-to-cytosol transport of CTA1. Derlin-1 interacts with CTB and the ER chaperone PDI as assessed by coimmunoprecipitation experiments. An in vitro membrane-binding assay showed that CTB stimulated the unfolded CTA1 chain to bind to the ER membrane. Moreover, intoxication of intact cells with CTB stabilized the degradation of a Derlin-1-dependent substrate, suggesting that CT uses the Derlin-1 pathway. These findings indicate that Derlin-1 facilitates the retro-translocation of CT. CTB may play a role in this process by targeting the holotoxin to Derlin-1, enabling the Derlin-1-bound PDI to unfold the A1 subunit and prepare it for transport.
霍乱毒素（CT）通过其受体结合B亚基（CTB）从质膜转运至内质网（ER）来使细胞中毒。在这个区室中，催化性A1亚基（CTA1）被蛋白二硫键异构酶（PDI）展开并逆向转运至胞质溶胶，在那里它触发信号级联反应，导致分泌性腹泻。目前尚不清楚CT如何靶向内质网膜中的逆向转运位点以启动转运。使用半透化细胞逆向转运试验，我们证明一种显性负性Derlin-1-YFP融合蛋白可减弱CTA1从内质网到胞质溶胶的转运。通过免疫共沉淀实验评估，Derlin-1与CTB和内质网伴侣蛋白PDI相互作用。体外膜结合试验表明，CTB刺激展开的CTA1链与内质网膜结合。此外，用CTB对完整细胞进行中毒处理可稳定Derlin-1依赖性底物的降解，这表明CT利用Derlin-途径。这些发现表明Derlin-1促进CT的逆向转运。CTB可能通过将全毒素靶向Derlin-1在这一过程中发挥作用，使与Derlin-1结合的PDI能够展开A1亚基并使其为转运做好准备。
