Abstract
Background Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae. Methodology and Principal FindingsUsing a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors.Conclusions and SignificanceThe fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.
Highlights
Endoplasmic reticulum (ER) associated protein degradation (ERAD) comprises multiple disposal pathways that recognize and remove terminally misfolded and orphan proteins from the endoplasmic reticulum (ER) membrane and lumen
The fraction of SLTxA1 that disengages from the ER-associated degradation (ERAD) pathway does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation
The dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus
Summary
Endoplasmic reticulum (ER) associated protein degradation (ERAD) comprises multiple disposal pathways that recognize and remove terminally misfolded and orphan proteins from the ER membrane and lumen. Removal (dislocation) normally requires specific membrane-bound ubiquitin ligase complexes that polyubiquitylate the target substrates [1,2], providing tags for an ERAD-enabled cytosolic AAA-ATPase p97/Cdc48p complex extraction motor [3,4,5,6] and, after limited deubiquitylation [7,8], for subsequent binding to proteasomes [9,10]. A number of proteins are thought to utilize ERAD components to reach the cytosol but disengage from canonical ERAD pathways and so avoid degradation in the proteasome core Principal amongst these are the enzymatic A chains of some plant and bacterial toxins which traffic in vesicular carriers from the cell surface of target mammalian cells to the ER lumen, where the toxic A chain and cell-binding B chain(s) are separated [12,13,14]. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae
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