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Membrane–cytosolic translocation of verotoxin A₁ subunit in target cells

¹ Research Institute, Program in Molecular Structure and Function, The Hospital for Sick Children, Ontario M5G 1X8, Canada
² Departments of Laboratory Medicine and Pathobiology, University of Toronto, Canada
³ Department of Biochemistry, University of Toronto, Canada

Correspondence
Clifford A. Lingwood
cling{at}sickkids.ca

In sensitive cells, verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [¹²⁵I]VT1-treated cells, the selective cytosolic translocation (4 %) of the activated form of VTA, VTA₁, was demonstrated for the first time by monitoring [¹²⁵I]VTA₁ release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic VTA₁ and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic VTA₁ detection. Brefeldin A and 16 °C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented VTA₁ cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA₁ requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic VTA₁ values indicates that about one molecule of translocated VTA₁ per cell is sufficient to inhibit protein synthesis and kill a cell.

Supplementary material showing quantitation of VT1 A₁ translocation and extrapolation to the CD₅₀ is available with the online version of this paper.