Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2
David E. Saslowsky, … , Barry H. Paw, Wayne I. Lencer
David E. Saslowsky, … , Barry H. Paw, Wayne I. Lencer
Published November 1, 2010
Citation Information: J Clin Invest. 2010;120(12):4399-4409. https://doi.org/10.1172/JCI42958.
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Research Article Cell biology

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2

Abstract

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft–associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex.

Authors

David E. Saslowsky, Jin Ah Cho, Himani Chinnapen, Ramiro H. Massol, Daniel J.-F. Chinnapen, Jessica S. Wagner, Heidi E. De Luca, Wendy Kam, Barry H. Paw, Wayne I. Lencer

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Figure 6

The flotillins affect sorting of CT into two pathways to the ER, only one intersecting the TGN.

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The flotillins affect sorting of CT into two pathways to the ER, only on...
(A) Cos-1 cells transfected with control or flotillin-1 siRNA were incubated with CT or CT plus CTB and protein extracts immunoblotted for CTA. (B) Cells transfected as in A were incubated with CT at 37°C for the indicated times. CTB was immunoprecipitated and immunoblotted using anti-CTB pAb. That no CTB was detected in lanes 3 and 4 indicates that virtually all the CTB was removed from the cell surface. Blank lane indicated by x. (C) Phosphorimage (top panels) of control or flotillin-1–depleted cells intoxicated for the indicated times with CTB-GS. Arrow indicates non-glycosylated CTB-GS and arrowhead the glycosylated form. A fraction of the post-IP eluate was analyzed for CTB by immunoblot (third panel). Intact CTB-GS is indicated by arrow (lower-MW forms are degradation products). (D) Immunoblot of post-ConA eluates from control or flotillin-1 siRNA cells intoxicated with CTB-GS. The glycosylated, higher-MW form is indicated by an arrowhead. Non-glycosylated CTB-GS is also present. (E) A fraction of the post-ConA eluates from D were analyzed by silver stain. (F) Phosphorimage of post-ConA eluates from Ctrl or flotillin-1 siRNA–treated cells preincubated with 35S-sulfate and subsequently intoxicated with CTB-GS. Arrow indicates non-glycosylated CTB-GS and arrowhead the glycosylated form. (G) Samples in F were also analyzed by silver stain. (H) Random, intoxicated cells were analyzed for the percentage of cells demonstrating colocalization of fluorescently labeled CTB and PDI at the NE (mean ± SEM; 6 independent experiments for Cos-1 cells and single experiments shown for Bsc-1 and A431 cells; number of cells imaged [n] is indicated). See Methods and Supplemental Video 3.