Vesicular Transport Is Not Required for the Cytoplasmic Pool of Cholera Toxin To Interact with the Stimulatory Alpha Subunit of the Heterotrimeric G Protein

Abstract

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic A1 polypeptide of CT (CTA1) then crosses the ER membrane, enters the cytosol, ADP-ribosylates the stimulatory ␣ subunit of the heterotrimeric G protein (Gs␣) at the cytoplasmic face of the plasma membrane, and activates adenylate cyclase. The cytosolic pool of CTA1 may reach the plasma membrane and its Gs␣ target by traveling on anterograde-directed transport vesicles. We examined this possibility with the use of a plasmid-based transfection system that directed newly synthesized CTA1 to either the ER lumen or the cytosol of CHO cells. Such a system allowed us to bypass the CT retrograde trafficking itinerary from the cell surface to the ER. Previous work has shown that the ER-localized pool of CTA1 is rapidly exported from the ER to the cytosol. Expression of CTA1 in either the ER or the cytosol led to the activation of Gs␣, and Gs␣ activation was not inhibited in transfected cells exposed to drugs that inhibit vesicular traffic. Thus, anterograde transport from the ER to the plasma membrane is not required for the cytotoxic action of CTA1.Vibrio cholerae produces cholera toxin (CT), a protein that induces the life-threatening diarrhea characteristic of cholera. CT ADP-ribosylates and irreversibly activates the stimulatory ␣-subunit of the heterotrimeric G protein (Gs␣) at the cytoplasmic face of the plasma membrane. Activated Gs␣ stimulates adenylate cyclase activity, and the subsequent increase in intracellular adenosine-3Ј,5Ј-monophosphate (cAMP) triggers chloride efflux from intoxicated intestinal epithelial cells. Diarrhea results from the osmotic movement of water that follows chloride efflux into the intestinal lumen (reviewed in reference 35).CT is an AB 5 -type protein toxin with a catalytic A subunit and a cell-binding B subunit (reviewed in reference 9). Proteolytic nicking of the A subunit at arginine 192 generates separate N-terminal A1 and C-terminal A2 fragments that remain linked by a disulfide bond between the cysteine residues at positions 187 and 199 in CTA. Catalytic activity resides in the CTA1 polypeptide, while the CTA2 polypeptide links CTA1 to CTB and stabilizes the holotoxin. The B subunit of CT is organized as a homopentameric ring-like structure that binds to GM1 gangliosides on the eukaryotic plasma membrane. Binding to GM1 gangliosides triggers toxin internalization from the cell surface through a caveolae or caveolae-like endocytic mechanism (1, 6, 30, 43). The active pool of internalized toxin subsequently moves from the endosomes to the Golgi and travels to the endoplasmic reticulum (ER) by retrograde vesicular transport (6,15,17,22,24,26,29). Reduction of the CTA1-CTA2 disulfide bond occurs in the ER, and a redox-dependent chaperone known as protein disulfide isomerase (PDI) catalyzes the release of CTA1 from CTA2/ CTB 5 (28, 40). The released CTA1 polypeptide then crosses the ER membrane, enters the cytosol, and initiates the major toxic effects of ...

Conformational Instability of the Cholera Toxin A1 Polypeptide

“…Inefficient degradation of CTA1 at 33°C was not due to an inhibitory effect on CTA1 export from the ER to the cytosol because the same temperature-dependent effects were observed for the turnover of a CTA1 construct (ΔssCTA1) that lacked the CTA signal sequence and was therefore expressed directly in the cytosol. 43 The attenuated degradation of ERAD substrate α1AT-Z in ts20 cells incubated at 41°C (in comparison to E36 cells incubated at 41°C) confirmed that ubiquitin-dependent processing events were inhibited at 41°C in the ts20 cells (Table I). Thus, the turnover of CTA1 was temperature-sensitive but ubiquitin-independent.…”

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

“…This differential affinity suggests a possible mechanism for CTA1 delivery to raft-localized G s ␣. The translocated pool of CTA1 appears to reach its G s ␣ target by diffusion through the cytosol (51). Binding to the plasma membrane would capture cytosolic CTA1 and place it in the general vicinity of G s ␣.…”

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

“…CTA1 Transfection Assays-Cells seeded to 80% confluency in 6-well plates were transfected with pcDNA3.1/ssCTA1 (35) using Lipofectamine (Invitrogen) according as per manufacturer's instructions. For the translocation assay, cells were incubated at 24 h post-transfection in methionine-free medium for 1 h before [ 35 S]methionine was added for another hour.…”

“…8B). A plasmid-based system was used to express CTA1 directly in the ER of transfected cells (35). cAMP levels were then quantified at 3 h post-transfection.…”