Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum

Abstract

Cholera toxin (CT), an AB(5)-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.

Keywords: ERAD; cholera toxin; lipid rafts; retro-translocation; retrograde pathway.

Figure 1

Current model for CT cell entry and intoxication. CT, via its B-subunit, binds to GM1 on the apical membrane of intestinal epithelial cells. It then traffics through early and recycling endosomes [2,3] to the TGN, perhaps bypassing the Golgi apparatus for eventual delivery to the ER [4,5]. From here, CT can recycle between the Golgi and ER (dotted arrows) [4]. Once inside the ER, the A1-chain is unfolded by PDI [1], recognized by the ER Hsp70 chaperone BiP (heavy chain binding protein) [6] and is presumably rendered a soluble substrate for retro-translocation by the ERAD-lumenal pathway [7]. Retro-translocation possibly involves the ER membrane proteins Hrd1, derlin-1 [8,9,10] and the Sec61 translocon [11]. Upon entry into the cytosol, the A1-chain refolds into its native conformation and activates adenylate cyclase (AC) by ADP-ribosylation of the hetero-trimeric G-protein Gs. The increase in cAMP causes chloride secretion and the massive diarrhea that typifies cholera [12].

Figure 2

Three-dimensional structure of CT [14,15]. The A-subunit non-covalently associates with the pentameric B-subunit. The A-subunit is further subdivided into A1- and A2-chains, which are separated by a protease cleavage site and are joined by a disulfide bond and further non-covalent interactions.

Figure 3

Retrograde Trafficking from the PM to the ER. CT binds to the ganglioside GM1 (blue) found in membrane microdomains (lipid rafts-green) on the plasma membrane of host cells, and can cluster five GM1 molecules at once. The toxin enters the cell by various endocytic mechanisms, including clathrin and caveolin-dependent, as well as caveolin and dynamin-independent mechanisms, and traffics to early and recycling endosomes [26,27,28,32]. Transport to the TGN involves many different proteins including V- and T-SNAREs [35,36]. From the TGN, the toxin traffics to the ER, apparently bypassing the Golgi-cisternae.A fraction of CT might be transported directly from endosome to ER. In the ER, the A1-chain is unfolded and retro-translocated to the cytosol.Available evidence indicates that the toxin B-subunit is bound to GM1 for the duration of the journey back to the ER.

Figure 4

Schematic of CT retro-translocation. It is still unclear exactly how retro-translocation proceeds. This figure shows the candidate proteins thought to be involved. Following entry into the ER, CT remains bound to GM1 and can interact with Derlin-1 [8,9] and Hrd1 [10]. PDI unfolds CT [1], while Erp72 works in opposition to maintain it in a folded conformation [45]. There is evidence that Sec61 is the retro-translocation channel [11], but these studies are not fully conclusive and Hrd1 and gp78 are also candidates [10] for forming the protein-conducting channel. Perhaps, Derlin-1, Hrd1, and Sec61 are located in close proximity and act together. Finally, upon entry into the cytosol the A1-chain might immediately bind Arf6 or another chaperone to refold rapidly into a stable enzymatically active conformation and to avoid rapid degradation by the 20S proteasome [48,51].