Disulfide Transfer between Two Conserved Cysteine Pairs Imparts Selectivity to Protein Oxidation by Ero1

Sevier, Carolyn S.; Kaiser, Chris A.

doi:10.1091/mbc.e05-05-0417

Cited by 65 publications

(62 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ero1␤ is functionally equivalent to Ero1p (21), and both Ero1␤ and Ero1p bind to PDI (31) and homodimerise (32). Since Ero1p and Ero1␤ also share conserved FAD binding residues (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1). However, it was possible that mutations in this region could cause conformational changes resulting in misfolding, particularly since Ero1p seems to expose buried residues during the oxidation cycle (32). To compare the overall conformation of wild-type Ero1␤ and the mutant proteins, we used a limited proteolysis approach.…”

Section: Resultsmentioning

confidence: 99%

“…EroEro interactions may therefore be important for Ero1␤ regulation and/or activity (32). To ask whether ero1-1 and ero1-2 phenotypes could be partly explained by a failure of mutant Eros to self-complex, we investigated whether the G252S and H254Y mutants interacted with wild-type Ero1␤.…”

Section: Differences In the Covalent And Non-covalent Interactions Ofmentioning

confidence: 99%

“…Recent work has suggested that Ero-Ero dimers contribute to Ero function (22,32). Given that an Ero1␤C396A mutant does not homodimerise efficiently, it was possible that mutations in the FAD binding domain also prevent dimerization.…”

Section: Differences In the Covalent And Non-covalent Interactions Ofmentioning

confidence: 99%

See 3 more Smart Citations

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

Dias-Gunasekara

Lith

Williams

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 Ero1␤ or the non-covalent interaction of Ero1␤ with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1␤ heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1␤ 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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Differences In the Covalent And Non-covalent Interactions Ofmentioning

confidence: 99%

Section: Differences In the Covalent And Non-covalent Interactions Ofmentioning

confidence: 99%

See 2 more Smart Citations

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

Dias-Gunasekara

Lith

Williams

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…It seems likely that in the cells Cys 30 -Cys 33 can shuttle electrons to Cys 130 -Cys 133 through both intersubunit and intermolecular reactions and maybe even via intrachain shuttle. Similarly, for Ero1p a mechanism of both intra-and interchain electron transfer has been suggested (34).…”

Section: Discussionmentioning

confidence: 96%

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Ang

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX 16 C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX 16 C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1-4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4 -6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the "small Tim" and Cox17 families (7-10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11-13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1␣ and Ero1␤) are large flavoenzymes present in the ER with...

show abstract

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Cain

Peters

Hailu

et al. 2013

Biotechnology Progress

View full text Add to dashboard Cite

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells but reported yields from Chinese hamster ovary (CHO) cells are lower at ∼300 mg/L. We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X-box binding protein (XBP-1S) and endoplasmic reticulum oxidoreductase (ERO1-Lα) and has been named CHOS-XE. CHOS-XE cells produced increased antibody (MAb) yields (5.3- 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L.

show abstract

Disulfide Transfer between Two Conserved Cysteine Pairs Imparts Selectivity to Protein Oxidation by Ero1

Cited by 65 publications

References 38 publications

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

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

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Contact Info

Product

Resources

About