Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

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Catalysis of Protein Folding by Protein Disulfide Isomerase and Small-Molecule Mimics

Antioxidants and Redox Signaling ◽

10.1089/152308603768295159 ◽

2003 ◽

Vol 5 (4) ◽

pp. 413-424 ◽

Cited By ~ 50

Author(s):

Elizabeth A. Kersteen ◽

Ronald T. Raines

Keyword(s):

Protein Folding ◽

Small Molecule ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Protein Disulfide

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Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011766 ◽

2020 ◽

Vol 295 (26) ◽

pp. 8647-8655 ◽

Cited By ~ 3

Author(s):

Antti Moilanen ◽

Lloyd W. Ruddock

Keyword(s):

Protein Folding ◽

Cross Talk ◽

Protein Disulfide Isomerase ◽

Feedback Inhibition ◽

Inhibition Mechanism ◽

Disulfide Isomerase ◽

Unfolded Protein ◽

Oxidative Protein Folding ◽

Native Proteins ◽

Protein Disulfide

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfide-isomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation–reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways.

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Protein disulfide isomerase activity is released by activated platelets

Blood ◽

10.1182/blood.v79.9.2226.2226 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2226-2228 ◽

Cited By ~ 77

Author(s):

K Chen ◽

Y Lin ◽

TC Detwiler

Keyword(s):

Disulfide Bonds ◽

Protein Disulfide Isomerase ◽

Ph Dependence ◽

Gel Filtration ◽

Disulfide Isomerase ◽

Isomerase Activity ◽

Activated Platelets ◽

Protein Disulfide ◽

Supernatant Solution ◽

Disulfide Isomerase Activity

Abstract The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin- thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

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