Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

Pathways for protein disulphide bond formation

The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems.     

Balanced Ero1 activation and inactivation establishes ER redox homeostasis

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.     

Modulation of cellular disulfide-bond formation and the ER redox environment by feedback regulation of Ero1

Introduction of disulfide bonds into proteins entering the secretory pathway is catalyzed by Ero1p, which generates disulfide bonds de novo, and Pdi1p, which transfers disulfides to substrate proteins. A sufficiently oxidizing environment must be maintained in the endoplasmic reticulum (ER) to allow for disulfide formation, but a pool of reduced thiols is needed for isomerization of incorrectly paired disulfides. We have found that hyperoxidation of the ER is prevented by attenuation of Ero1p activity through noncatalytic cysteine pairs. Deregulated Ero1p mutants lacking certain cysteines show increased enzyme activity, a decreased lag phase in kinetic assays, and growth defects in vivo. We hypothesize that noncatalytic cysteine pairs in Ero1p sense the level of potential substrates in the ER and correspondingly modulate Ero1p activity as part of a homeostatic regulatory system governing the thiol-disulfide balance in the ER.     

Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum

In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol-disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol-disulfide exchange with ER oxidoreductases. The Cys352-Cys355 disulfide could then serve to reoxidize the Cys100-Cys105 cysteine pair, possibly through an intramolecular thiol-disulfide exchange reaction.     

Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.     

Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum.

Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p. Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI. PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant. Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides. Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY). Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo. Together, these results define a pathway for protein disulfide bond formation in the ER. The PDI homolog Mpd2p is also oxidized by Ero1p.     

Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.     

The prokaryotic enzyme DsbB may share key structural features with eukaryotic disulfide bond forming oxidoreductases

Three different classes of thiol-oxidoreductases that facilitate the formation of protein disulfide bonds have been identified. They are the Ero1 and SOX/ALR family members in eukaryotic cells, and the DsbB family members in prokaryotic cells. These enzymes transfer oxidizing potential to the proteins PDI or DsbA, which are responsible for directly introducing disulfide bonds into substrate proteins during oxidative protein folding in eukaryotes and prokaryotes, respectively. A comparison of the recent X-ray crystal structure of Ero1 with the previously solved structure of the SOX/ALR family member Erv2 reveals that, despite a lack of primary sequence homology between Ero1 and Erv2, the core catalytic domains of these two proteins share a remarkable structural similarity. Our search of the DsbB protein sequence for features found in the Ero1 and Erv2 structures leads us to propose that, in a fascinating example of structural convergence, the catalytic core of this integral membrane protein may resemble the soluble catalytic domain of Ero1 and Erv2. Our analysis of DsbB also identified two new groups of DsbB proteins that, based on sequence homology, may also possess a catalytic core similar in structure to the catalytic domains of Ero1 and Erv2.     

Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell

The flavoenzyme Ero1p produces disulfide bonds for oxidative protein folding in the endoplasmic reticulum. Disulfides generated de novo within Ero1p are transferred to protein disulfide isomerase and then to substrate proteins by dithiol-disulfide exchange reactions. Despite this key role of Ero1p, little is known about the mechanism by which this enzyme catalyzes thiol oxidation. Here, we present the X-ray crystallographic structure of Ero1p, which reveals the molecular details of the catalytic center, the role of a CXXCXXC motif, and the spatial relationship between functionally significant cysteines and the bound cofactor. Remarkably, the Ero1p active site closely resembles that of the versatile thiol oxidase module of Erv2p, a protein with no sequence homology to Ero1p. Furthermore, both Ero1p and Erv2p display essential dicysteine motifs on mobile polypeptide segments, suggesting that shuttling electrons to a rigid active site using a flexible strand is a fundamental feature of disulfide-generating flavoenzymes.     

Reexamination of the role of interplay between glutathione and protein disulfide isomerase

Protein disulfide isomerase (PDI) has an essential role in the process of disulfide bond formation, where it catalyzes disulfide bond formation, reduction, and isomerization. It is thought that the major route for oxidizing dithiols in folding proteins to disulfides is via Ero1-mediated oxidation of PDI. Since the discovery of Ero1, the role of glutathione in disulfide bond formation has been downplayed. In this study, the role of glutathione in disulfide bond formation was reexamined. Here we have studied in vitro the kinetics of the glutathione-mediated oxidation and reduction of the catalytic a domains of human PDI and yeast Pdi1p. The results obtained from stopped-flow and quenched-flow experiments show that the reactions of PDI and Pdi1p are faster and more complex than previously thought. Our results suggest that the kinetics of oxidation of PDI and Pdi1p by oxidized glutathione are remarkably similar, whereas the kinetics of reduction by reduced glutathione shows clear differences. The data generated here on the rapid reactivity of PDI towards glutathione suggest that reevaluation is required for several aspects of the field of catalyzed disulfide bond formation, including the potential physiological role of glutathione.     

Disulfide relays between and within proteins: the Ero1p structure

The essential flavoenzyme Ero1p both creates de novo disulfide bonds and transfers these disulfides to the folding catalyst protein disulfide isomerase (PDI). The recently solved crystal structure of Ero1p, in combination with previous biochemical, genetic and structural data, provides insight into the mechanism by which Ero1p accomplishes these tasks. A comparison of Ero1p with the smaller flavoenzyme Erv2p highlights important structural elements that are shared by these flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases and suggests some general themes that might be common to proteins that generate disulfide bonds.     

The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha

The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397). Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.     

Steps in reductive activation of the disulfide-generating enzyme Ero1p

Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity.     

A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.     

Competition between glutathione and protein thiols for disulphide-bond formation

It has long been assumed that the oxidized form of glutathione, the tripeptide glutamate-cysteine-glycine, is a source of oxidizing equivalents needed for the formation of disulphide bonds in proteins within the endoplasmic reticulum (ER), although the in vivo function of glutathione in the ER has never been studied directly. Here we show that the major pathway for oxidation in the yeast ER, defined by the protein Ero1, is responsible for the oxidation of both glutathione and protein thiols. However, mutation and overexpression studies show that glutathione competes with protein thiols for the oxidizing machinery. Thus, contrary to expectation, cellular glutathione contributes net reducing equivalents to the ER; these reducing equivalents can buffer the ER against transient hyperoxidizing conditions.     

Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum

Disulphide formation in the endoplasmic reticulum (ER) is catalysed by members of the protein disulphide isomerase (PDI) family. These enzymes can be oxidized by the flavoprotein ER oxidoreductin 1 (Ero1), which couples disulphide formation with reduction of oxygen to form hydrogen peroxide (H(2)O(2)). The H(2)O(2) produced can be metabolized by ER-localized peroxiredoxin IV (PrxIV). Continuous catalytic activity of PrxIV depends on reduction of a disulphide within the active site to form a free thiol, which can then react with H(2)O(2). Here, we demonstrate that several members of the PDI family are able to directly reduce this PrxIV disulphide and in the process become oxidized. Furthermore, we show that altering cellular expression of these proteins within the ER influences the efficiency with which PrxIV can be recycled. The oxidation of PDI family members by PrxIV is a highly efficient process and demonstrates how oxidation by H(2)O(2) can be coupled to disulphide formation. Oxidation of PDI by PrxIV may therefore increase efficiency of disulphide formation by Ero1 and also allows disulphide formation via alternative sources of H(2)O(2).     

Erv14p directs a transmembrane secretory protein into COPII-coated transport vesicles

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.     

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.