N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Abstract

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.Like most toxins, cholera toxin (CT) 3 must cross a cell membrane to enter the cytosol of host cells and induce disease. To do this, CT traffics retrograde from the plasma membrane to the endoplasmic reticulum (ER), where it co-opts the machinery for degradation of terminally misfolded proteins in the ER (1). Unlike the misfolded substrates, however, the toxin escapes rapid degradation by the proteasome and refolds to its native conformation so as to act enzymatically in the cytosol (2). This is evidenced most clearly by the lack of sensitivity to chemical inhibition of the proteasome (3). How the A1-chain avoids rapid degradation to induce toxicity, however, is not fully understood. The prevailing view is that the paucity of lysines explains the resistance to ubiquitination and proteasomal degradation (4). Another view is that cytosolic chaperones stabilize the fold of the A1-chain so as to prevent non-ubiquitin-dependent degradation by the 20S proteasome, but degradation of the A1-chain in vivo is slow, occurring after the induction of toxicity (5).CT typifies the AB 5 family of toxins and is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. The A-subunit is comprised of an enzymatically active A1-chain linked non-covalently to the B-subunit via the A2-chain. The A1-and A2-chains are joined by a flexible loop containing a serine-protease cleavage site bridged by a disulfide bond. Both bonds must be broken before the A1-chain can enter the cytosol of host cells (2). The B subunit consists of five 11.5-kDa peptides assembled non-covalently into a stable homopentamer that binds to the ganglioside GM1 on the plasma membrane. The B-subunit-GM1 complex carries the A-subunit into the ER (6).In the ER, protein disulfide isomerase (PDI) in its reduced state unfolds and dissociates the A1-chain from the B-subunit. When oxidized, PDI releases the A1-chain to subsequent steps in the retrotranslocation reaction (...

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

“…Therefore, we assayed for effects of Derlin-1 on retro-translocation of the A1-chain, measured as the presence of the A1-chain in cytosolic fractions of cells by immunoblot (methods modified from refs. 26,35). The results show similar or even slightly elevated levels of A1-chain in the cytosolic fractions of cells lacking Derlin-1 ( Figure 3E, upper left panel, compare lanes 2 and 3 with lane 1).…”

“…Depletion of flotillin-1 and -2 caused a small decrease in 35 S-sulfation of CTB-GS after 40 and 120 minutes of toxin uptake, indicating a very modest reduction (between 9% and 30%) in transport from PM into the TGN (Figure 6C, arrow). At 120 minutes, a higher-MW 35 S band was also evident (arrowhead, lanes 3 and 4), representing the ER N-glycosylated form of CTB-GS as confirmed by cleavage with the endoglycosidase PNGase F (lanes 5 and 6). TCA precipitation of total protein extracts showed that 35 S-sulfotransferase activity was unaffected by depletion of flotillin-1 and -2 (Supplemental Figure 6A).…”

“…To test whether toxin action in zebrafish requires transport into the ER and retro-translocation as in mammalian cells, we utilized G33D and the mutant toxin R192G, which contains a WT B-subunit but a mutated A-subunit that cannot unfold in the ER or retro-translocate (20,35). Only the WT toxins induced toxicity in embryos from 3 different fish strains ( Figure 2C, asterisks).…”

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…CTxA1 into a bona-fide ERAD substrate (Wernick et al 2010). Thus, dislocation and refolding of the wild-type toxic polypeptides must proceed in a way that protects canonical sites for ubiquitylation from the Hrd1 ubiquitin ligase activity.…”

“…CTx binds both gp78 and Hrd1 and since these are E3 ubiquitin ligases, a role for ubiquitin in dislocation has been proposed (Bernardi et al 2010). However, like RTA, wild-type CTxA dislocates in a manner independent of canonical ubiquitylation (Rodighiero et al 2002) and N-terminal extension of CTxA results in its conversion to an ERAD substrate by displacing the two lysyl residues normally near the N terminus, making them substrates for dislocation-associated polyubiquitylation (Wernick et al 2010). An initial view that Derlin-1 is required for CTxA1 dislocation (Bernardi et al 2008) has been reversed after taking into account the effect of manipulating Derlin-1 levels in the absence of CTx challenge (Saslowsky et al 2010).…”