title: 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 Gsα 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.
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32826, USA.
authors.forename: Shane
authors.initials: S
authors.lastname: Massey
authors.forename: Helen
authors.initials: H
authors.lastname: Burress
authors.forename: Michael
authors.initials: M
authors.lastname: Taylor
authors.forename: Kathleen N
authors.initials: KN
authors.lastname: Nemec
authors.forename: Supriyo
authors.initials: S
authors.lastname: Ray
authors.forename: David B
authors.initials: DB
authors.lastname: Haslam
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D000818:Animals; D016466:CHO Cells; D002772:Cholera Toxin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D011485:Protein Binding; D017510:Protein Folding
chemical_list: C494648:DNAJB11 protein, human; D050956:HSP40 Heat-Shock Proteins; D002772:Cholera Toxin