Mutations in the FAD Binding Domain Cause Stress-induced Misoxidation of the Endoplasmic Reticulum Oxidoreductase Ero1β

Sanjika Dias-Gunasekara,Marcel Van Lith,J A Gareth Williams,Ritu Kataky,Adam M Benham

doi:10.1074/jbc.m602354200

Sanjika Dias-Gunasekara, Marcel Van Lith + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m602354200

Copy DOI

Journal: The Journal of biological chemistry	Publication Date: Sep 1, 2006
Citations: 9	License type: cc-by

Affiliation: Durham University

Abstract

Disulfide bond catalysis is an essential component of protein biogenesis in the secretory pathway, from yeast through to man. In the endoplasmic reticulum (ER), protein-disulfide isomerase (PDI) catalyzes the oxidation and isomerization of disulfide bonds and is re-oxidized by an endoplasmic reticulum oxidoreductase (ERO). The elucidation of ERO function was greatly aided by the genetic analysis of two ero mutants, whose impairment results from point mutations in the FAD binding domain of the Ero protein. The ero1-1 and ero1-2 yeast strains have conditional and dithiothreitol-sensitive phenotypes, but the effects of the mutations on the behavior of Ero proteins has not been reported. Here, we show that these Gly to Ser and His to Tyr mutations do not prevent the dimerization of Ero1beta or the non-covalent interaction of Ero1beta with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1beta heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1beta susceptible to misoxidation and aggregation, particularly during a temperature or redox stress. We conclude that the Ero FAD binding domain is critical for conformational stability, allowing Ero proteins to withstand stress conditions that cause client proteins to misfold.

Highlights

Cytosol by the proteasome [2]
Ero1␤ is functionally equivalent to Ero1p [21], and both Ero1␤ and Ero1p bind to protein-disulfide isomerase (PDI) [31] and homodimerise [32]
Since Ero1p and Ero1␤ share conserved FAD binding residues (Fig. 1A), we used our knowledge of Ero1␤ to study the effects of the ero1-1 and ero1-2 mutations on Endoplasmic reticulum oxidoreductases (Eros) gene products

Summary

Introduction

Cytosol by the proteasome [2]. Neurological disorders such as Alzheimer Disease, Huntington Chorea, and Creutzfeldt-Jakob disease all have an ER misfolding or stress component, and in amyloidosis, the cell specific ER environment and folding “signature” can determine whether a protein is appropriately secreted [3]. Transfected HeLa cell post-nuclear supernatants were treated with a concentration range of TPCK-trypsin for 30 min on ice. The reaction was quenched with soybean trypsin inhibitor and the lysates were analyzed by reducing SDS-PAGE, Western blotting, and detection with either ␣Myc or ␣Ero1␤. Similar results were obtained with the H254Y mutant: the single and double transfectants expressed the proteins expected (Fig. 3C, lanes 1–3) and the H254Y mutant co-immunoprecipitated with HAtagged wild-type Ero1␤ (Fig. 3D, lane 3).

Results

Conclusion