Functional in Vitro Analysis of the ERO1 Protein and Protein-disulfide Isomerase Pathway

Kazutaka Araki,Kazuhiro Nagata

doi:10.1074/jbc.m111.227181

Kazutaka Araki, Kazuhiro Nagata

Open Access

https://doi.org/10.1074/jbc.m111.227181

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Abstract

Oxidative protein folding in the endoplasmic reticulum is supported by efficient electron relays driven by enzymatic reactions centering on the ERO1-protein-disulfide isomerase (PDI) pathway. A controlled in vitro oxygen consumption assay was carried out to analyze the ERO1-PDI reaction. The results showed the pH-dependent oxidation of PDI by ERO1α. Among several possible disulfide bonds regulating ERO1α activity, Cys(94)-Cys(131) and Cys(99)-Cys(104) disulfide bonds are dominant regulators by excluding the involvement of the Cys(85)-Cys(391) disulfide in the regulation. The fine-tuned species specificity of the ERO1-PDI pathway was demonstrated by functional in vitro complementation assays using yeast and mammalian oxidoreductases. Finally, the results provide experimental evidence for the intramolecular electron transfer from the a domain to the a' domain within PDI during its oxidation by ERO1α.

Highlights

Protein disulfide isomerase (PDI) is a representative Endoplasmic reticulum (ER)-resident oxidoreductase that catalyzes disulfide formation and acts as a molecular chaperone [5, 6]
ERO1␣(C85A/ C104A/C131A/C391A) decreased the accelerated activity of ERO1␣(C104A/C131A) (Fig. 2D). These results suggest that its structure would not be disturbed by these mutations, because its oxygen consumption rate in the presence of DTT and its binding affinity for PDI were identical to those of ERO1␣(WT) (Fig. 2, E and F). These results demonstrate that Cys94–Cys104 and Cys99–Cys131 disulfide bonds serve a major negative regulatory function and that Cys85–Cys391 disulfide bond formation would be necessary for proper oxidative activity of ERO1␣
The oxygen consumption assay is used in the analysis of the ERO1 oxidase activity

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Human PDI and ERO1␣ cDNAs without the signal sequence were amplified by PCR from a Matchmaker Pretransformed Human HeLa library (Clontech) and subcloned into the modified pETDuet-1 vector (Novagen) modified with an N-terminal His tag and TEV (tobacco etch virus) protease recognition site called the pETDuet-TEV vector. The eluted ERO1␣ sample was oxidized with the addition of potassium ferricyanide at a final concentration of 20 mM for 20 min on ice, which was followed by gel-permeation chromatography (HiLoad 16/60 Superdex 200-pg column, GE Healthcare) pre-equilibrated with 20 mM HEPES-NaOH (pH 7.4) containing 150 mM NaCl, 10% glycerol, and no imidazole. SPR Measurements—Association or dissociation rate constants (kon or koff) for the direct binding of PDI/Pdi1p or their variants to immobilized ERO1␣(WT) or its variants were determined by surface plasmon resonance (SPR) measurements on a ProteOn XPR36 Protein Interaction Array System (Bio-Rad). Sensorgrams were recorded simultaneously for five concentrations of analytes ranging from 0.44 ␮M to 36 ␮M in triplicate increments at 25 °C for a 2-min association phase, followed by a 10-min dissociation phase by using 20 mM HEPES-NaOH (pH 7.5; except Fig. 1D), 150 mM NaCl, 0.001% Tween 20 and 2 mM EDTA as running and sample buffer.

RESULTS

DISCUSSION

Kazutaka Araki and Kazuhiro Nagata