Cholera toxin (CT), an AB5-subunit toxin, enters host cells by binding the ganglioside GM1 at the plasma membrane (PM) and travels retrograde through the trans-Golgi Network into the endoplasmic reticulum (ER). In the ER, a portion of CT, the enzymatic A1-chain, is unfolded by protein disulfide isomerase and retro-translocated to the cytosol by hijacking components of the ER associated degradation pathway for misfolded proteins. After crossing the ER membrane, the A1-chain refolds in the cytosol and escapes rapid degradation by the proteasome to induce disease by ADP-ribosylating the large G-protein Gs and activating adenylyl cyclase. Here, we review the mechanisms of toxin trafficking by GM1 and retro-translocation of the A1-chain to the cytosol.
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 (...
Endocytosis of membrane proteins is typically mediated by signals present in their cytoplasmic domains. These signals usually contain an essential tyrosine or pair of leucine residues. Both tyrosine-and dileucinebased endocytosis signals are recognized by the adaptor complex AP-2. The best understood of these interactions occurs between the tyrosine-based motif, YXX⌽, and the 2 subunit of AP-2. We recently reported a tryptophanbased endocytosis signal, WLSL, contained within the cytoplasmic domain of the neonatal Fc receptor. This signal resembles YXX⌽. We have investigated the mechanism by which the tryptophan-based signal is recognized. Both interaction assays in vitro and endocytosis assays in vivo show that 2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXX⌽. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the ␣ subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXX⌽ motif, and an unexpected versatility of 2 function.
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