Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2
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
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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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 5

Flotillins are required for CT function in mammalian cells.

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Flotillins are required for CT function in mammalian cells. (A) Cos-1 ce...
(A) Cos-1 cells were transfected with control or flotillin-1 (Flo-1) siRNA, and knockdown was gauged by immunoblot of crude cell lysates using antibodies against flotillin-1 and -2. (B) Cells treated as in A were incubated with buffer (no toxin) or 1 nM CT for the indicated times prior to analysis of intracellular cAMP by ELISA. Data (mean ± SEM; representative of 3 independent experiments) were normalized to forskolin response (~20% less forskolin-induced cAMP in flotillin-1 sRNA–treated cells as compared with controls; see text). (C) Cells transfected with control and flotillin-1 siRNA were incubated with 20 nM CT holotoxin for 90 minutes, and A1-chain retro-translocation was measured as in Figure 3E, except that a combination of CTA and CTB antibodies was used for immunoblot analysis (upper panel).