Glutathione Directly Reduces an Oxidoreductase in the Endoplasmic Reticulum of Mammalian Cells

Catherine E Jessop,Neil J Bulleid

doi:10.1074/jbc.m411409200

Abstract

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.

Highlights

In 1963 Anfinsen showed that the folding of a protein, ribonuclease, is spontaneous and depends entirely on the primary structure of the protein [1]
ERp57 Is Maintained in a Reduced State in Vivo—To assess the requirement for a reductive pathway, we first examined the redox state of several endoplasmic reticulum (ER) oxidoreductases within mammalian cells grown in culture
When the redox state of the known ER oxidoreductases ERp57, ERp72, P5, protein-disulfide isomerase (PDI)-related protein, and PDI was assessed using this approach, a clear decrease in mobility was seen for the proteins from intact cells treated with the oxidizing agent DPS in comparison with cells treated with the reducing agent DTT (Fig. 1A, lanes 1 and 2)

Summary

Introduction

In 1963 Anfinsen showed that the folding of a protein, ribonuclease, is spontaneous and depends entirely on the primary structure of the protein [1]. The oxidative pathway for disulfide bond formation has been well characterized where PDI is known to oxidize substrate proteins and is itself maintained in an oxidized form by Ero1 [5, 6], for which the ultimate electron acceptor can be molecular oxygen [7]. Other oxidoreductases such as ERp57 are thought to act as reductases or isomerases and must be maintained in a reduced state to remain active. This is the sole function of DsbD and given the similarities between the prokaryotic and eukaryotic oxidative pathways, it has often been speculated that pathways parallel to the prokaryotic reductive pathway may exist in eukaryotic cells

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