title: Conformational instability of the cholera toxin A1 polypeptide.
abstract: Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by vesicular transport. In the ER, the catalytic CTA1 subunit dissociates from the holotoxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). It is hypothesized that CTA1 triggers its ERAD-mediated translocation into the cytosol by masquerading as a misfolded protein, but the process by which CTA1 activates the ERAD system remains unknown. Here, we directly assess the thermal stability of the isolated CTA1 polypeptide by biophysical and biochemical methods and correlate its temperature-dependent conformational state with susceptibility to degradation by the 20S proteasome. Measurements with circular dichroism and fluorescence spectroscopy demonstrated that CTA1 is a thermally unstable protein with a disordered tertiary structure and a disturbed secondary structure at 37 degrees C. A protease sensitivity assay likewise detected the temperature-induced loss of native CTA1 structure. This protease-sensitive conformation was not apparent when CTA1 remained covalently associated with the CTA2 subunit. Thermal instability in the dissociated CTA1 polypeptide could thus allow it to appear as a misfolded protein for ERAD-mediated export to the cytosol. In vitro, the disturbed conformation of CTA1 at 37 degrees C rendered it susceptible to ubiquitin-independent degradation by the core 20S proteasome. In vivo, CTA1 was also susceptible to degradation by a ubiquitin-independent proteasomal mechanism. ADP-ribosylation factor 6, a cytosolic eukaryotic protein that enhances the enzymatic activity of CTA1, stabilized the heat-labile conformation of CTA1 and protected it from in vitro degradation by the 20S proteasome. Thermal instability in the reduced CTA1 polypeptide has not been reported before, yet both the translocation and degradation of CTA1 may depend upon this physical property.
authors.affiliation: Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32826, USA.
authors.forename: Abhay H
authors.initials: AH
authors.lastname: Pande
authors.forename: Patricia
authors.initials: P
authors.lastname: Scaglione
authors.forename: Michael
authors.initials: M
authors.lastname: Taylor
authors.forename: Kathleen N
authors.initials: KN
authors.lastname: Nemec
authors.forename: Summer
authors.initials: S
authors.lastname: Tuthill
authors.forename: David
authors.initials: D
authors.lastname: Moe
authors.forename: Randall K
authors.initials: RK
authors.lastname: Holmes
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002772:Cholera Toxin / Q000737:chemistry*; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004795:Enzyme Stability; D006358:Hot Temperature; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D000089982:ADP-Ribosylation Factor 6; D021122:Protein Subunits; D002772:Cholera Toxin; D046988:Proteasome Endopeptidase Complex; D020727:ADP-Ribosylation Factors