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Vol. 17, Issue 3, 1375-1387, March 2006

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The Association of Shiga-like Toxin with Detergent-resistant Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the Endoplasmic Reticulum for a Cytotoxic Effect

Daniel C. Smith ^* , Daniel J. Sillence  , Thomas Falguières  ^||, Rosemary M. Jarvis ^*, Ludger Johannes , J. Michael Lord ^*, Frances M. Platt , and Lynne M. Roberts ^*

^* Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; and Unité Mixte de Recherche 144 Curie/Centre National de la Recherche Scientifique, Traffic and Signalling Laboratory, Curie Institute, 75248 Paris Cedex 05, France

Submitted November 10, 2005; Revised December 7, 2005; Accepted December 20, 2005
Monitoring Editor: Sandra Schmid

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb₃) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.

Abbreviations used: CTx, cholera toxin; CsA, cyclosporine A; DRM, detergent-resistant membrane(s); ER, endoplasmic reticulum; Gb₃, globotriaosylceramide; GlcCer, glucosylceramide; GlcSph, glucosylsphingosine; GSL, glycosphingolipids; LacCer, lactosylceramide; NB-DGJ, N-butyldeoxygalactonojirimycin; STx, Shiga toxin; SLTx, Shiga-like toxin; SLTxB, Shiga-like toxin B-subunit; GM3, II³--N-acetylneuraminyllactosylceramide, nLc₄, neolactotetraglycosylceramide; Sialyl-nLc₄, IV³--N-acetylneuraminylneolactotetraglycosylceramide.

These authors contributed equally to this study.

^|| Present address: Department of Biochemistry, Sciences II, University of Geneva, 30 quai Ernest Ansermet, 1211 Geneva 4, Switzerland.

Address correspondence to: Lynne M. Roberts (lynne.roberts{at}warwick.ac.uk).

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