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 3

Derlin-1 is dispensable for CT function in zebrafish embryos and retro-translocation of the A1-chain in mammalian cells.

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Derlin-1 is dispensable for CT function in zebrafish embryos and retro-t...
(A) Protein extracts from 5 controls (Ctrl) or Derlin-1 morphants (72-hpf) were immunoblotted for Derlin-1 and β-actin. (B) Control or Derlin-1 morphants were intoxicated or not with 1.5 nM CT at 16- to 18-hpf and imaged at 72 hpf. Insets to scale with top panels. (C) Protein extracts from Cos-1 cells transfected with the indicated siRNA (Der1* indicates combination of 2 siRNAs) were subject to immunoblot as in A. (D) qRT-PCR for DERL1 from control and Der1 siRNA–treated cells (mean ± SD, n = 3). (E) Cytosolic and membrane fractions were prepared from Cos-1 cells transfected with the indicated siRNA and intoxicated with WT or R192G CT. Immunoblots were probed with CTA, CTB, BiP, or β-actin antibodies. Purified toxin standards, A1-chain, and uncleaved A1/A2-chain complex are indicated. (F) MFI of GFP expression in Cos-1 cells transfected with CFTRΔF508-GFP and control or Der1 siRNA. Maximal CFTRΔF508-GFP accumulation is achieved by treatment with the proteasome inhibitor MG132 (10 μM; Ctrl + MG132), and these values were set to 100% in each experiment. Blue bars indicate means, and error bar indicates variance of Ctrl + MG132 samples. (G) Immunoblot of crude protein extracts from samples in F probed with mAbs against CFTR and β-actin. Filled and open arrowheads represent the immature and mature glycosylated forms of CFTR, respectively. (H) Cells transfected with control, Derlin-2 (Der2), or a combination of Derlin-1 and Derlin-2 siRNA were analyzed for Derlin-1 or -2 by immunoblot. (I) Same as E, except cells were transfected with control, Derlin-2, or both Derlin-1 and -2 siRNA.