The Reduction Potential of the Active Site Disulfides of Human Protein Disulfide Isomerase Limits Oxidation of the Enzyme by Ero1α

Joseph E Chambers,Timothy J Tavender,Ojore B.V Oka,Stacey Warwood,David Knight,Neil J Bulleid

doi:10.1074/jbc.m110.156596

Abstract

Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.

Highlights

The process of disulfide formation occurring within secretory proteins entering the endoplasmic reticulum requires the combined activities of a protein disulfide isomerase (PDI)3 and Ero1 [1]
It has been shown that the minimal requirement for PDI to be oxidized by Ero1␣ is the presence of an active site domain C-terminal to the bЈ domain, suggesting that the specificity of oxidation of PDI by Ero1␣ lies with the positioning of the active site domains [12]
The results show that the intrinsic activity of Ero1␣ toward PDI in vitro is limited by the reduction potential of the PDI active sites

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Mutagenesis—Constructs expressing His-tagged PDI and ⌬S1 and ⌬S2 mutants were obtained from Lloyd Ruddock (Oulu, Finland). Gel slices were incubated with 50 mM 1,2-13C2-bromoacetic acid (Cambridge Isotopes), in 100 mM ammonium bicarbonate at 37 °C for 2 h in the dark. Mutant PDI gel slices were rehydrated with 5 ng/␮l trypsin and 10 ng/␮l Glu-C (Sigma) in 100 mM Tris-HCl buffer, pH 8.5, and incubated overnight at 37 °C. TCAprecipitated proteins were washed twice with ice-cold acetone and resuspended in 200 mM Tris-HCl buffer, pH 8 containing 6 M urea, 0.5% (w/v) SDS, and 50 mM iodoacetic acid and incubated at 37 °C for 2 h in the dark. If the area under the first isotopic peak ϭ r and the normalized area under the oxidized active site peptide peak ϭ ox, the fraction of reduced PDI peptide in each sample was defined as r/(r ϩ ox). (RT/nF) ln(Keq)) using the calculated Keq from Equation 1, T ϭ 25 °C, n ϭ 2, and the standard reduction potential of GSH ϭ Ϫ240 mV

RESULTS

WT PDI a domain

DISCUSSION