Structural and Functional Interactions between the Cholera Toxin A1 Subunit and ERdj3/HEDJ, a Chaperone of the Endoplasmic Reticulum

Abstract

Cholera toxin (CT) is endocytosed and transported by vesicle carriers to the endoplasmic reticulum (ER).The catalytic CTA1 subunit then crosses the ER membrane and enters the cytosol, where it interacts with its Gs␣ target. The CTA1 membrane transversal involves the ER chaperone BiP, but few other host proteins involved with CTA1 translocation are known. BiP function is regulated by ERdj3, an ER-localized Hsp40 chaperone also known as HEDJ. ERdj3 can also influence protein folding and translocation by direct substrate binding. In this work, structural and functional assays were used to examine the putative interaction between ERdj3 and CTA1. Cell-based assays demonstrated that expression of a dominant negative ERdj3 blocks CTA1 translocation into the cytosol and CT intoxication. Binding assays with surface plasmon resonance demonstrated that monomeric ERdj3 interacts directly with CTA1. This interaction involved the A1 2 subdomain of CTA1 and was further dependent upon the overall structure of CTA1: ERdj3 bound to unfolded but not folded conformations of the isolated CTA1 subunit. This was consistent with the chaperone function of ERdj3, as was the ability of ERdj3 to mask the solvent-exposed hydrophobic residues of CTA1. Our data identify ERdj3 as a host protein involved with the CT intoxication process and provide new molecular details regarding CTA1-chaperone interactions.Many toxins share a structural organization that consists of a catalytic A subunit and a cell-binding B subunit (32). These extracellular toxins attack targets within the eukaryotic cytosol and must therefore cross a membrane barrier in order to function. Some AB toxins, such as diphtheria toxin (DT), access the cytosol from acidified endosomes. Other AB toxins move from the plasma membrane to the endoplasmic reticulum (ER) before passage into the cytosol by a process involving the quality control system of ER-associated degradation (ERAD) (14, 21). For both endosome and ER translocation sites, holotoxin disassembly precedes or occurs concurrently with A chain entry into the cytosol.The process of ERAD-mediated toxin translocation is not completely defined, but some details have been elucidated for ERAD interactions with the catalytic A1 subunit of cholera toxin (CTA1). The single disulfide bond linking CTA1 to the rest of the cholera holotoxin (CT) is reduced at the resident redox state of the ER (22). Reduced CTA1 dissociates from the holotoxin with the aid of protein disulfide isomerase (PDI) (39, 45), and unfolding of the isolated CTA1 polypeptide then facilitates its passage into the cytosol through the Sec61 and/or Derlin-1 protein-conducting channels (5,11,33,34). Most ERAD substrates are efficiently degraded in the cytosol by the ubiquitin-proteasome system (47). CTA1 avoids this fate because it only has two lysine residues to serve as potential ubiquitin attachment sites (14,30,31). The translocated pool of CTA1 thus persists in the cytosol long enough to modify its Gs␣ target. A hydrophobic region in the C-terminal A1 3 subdomain o...

Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

“…From these results we concluded that CTA1 has partially disordered secondary structure and can be placed in the intrinsically disordered (ID) protein class. The behavior of cholera toxin A1 polypeptide of regular units of helices and β-sheets interrupted by coils were in close agreement to the work of Ampapathi et al [16] who observed the secondary structure through circular dichroism spectroscopy and 2D nuclear magnetic resonance spectroscopy and that of others [13][14][15][16].…”

“…The A-subunit (CT-A, MW ~ 27,400) is the enzymatic domain [10,11], it contains 240 amino acid residues. The first 132 amino acids formed a compact globular unit composed of a mixture of α-helices and β-strands [10,12,13]. The catalysis is thought to occur in well define fissure on the free surface of A-subunit [10,12,14].…”

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

“…52. After establishment of a baseline measurement (0 refractive index unit; RIU) corresponding to the mass of the sensor-bound CTA1, Hsp90, ARF6, or other ligands were perfused over the CTA1-coated sensor slide.…”

A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40

“…While a J-protein called ERdj3 has been implicated in SV40 infection (5), NEFs have yet to be demonstrated to be involved in this process. In fact, more generally, very little is known regarding NEF functions during ERAD (21), in contrast to J-protein functions (22)(23)(24)(25).…”