Ero1p Oxidizes Protein Disulfide Isomerase in a Pathway for Disulfide Bond Formation in the Endoplasmic Reticulum
ABSTRACT 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.
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- "Here, the thiol group can undergo reversible oxidation into sulfenic acid or irreversible oxidation into sulfinic-or sulfonic acid; the thiol group can also be S-glutathionylated or, if reactive nitrogen species are present, nitrosylated. Proteins undergoing oxidative folding within the ER gain disulfides by dithiol-disulfide exchange with the oxidized form of the thioredoxin-like protein Pdi1p (Frand & Kaiser, 1999; Tu & Weissman, 2004). A shift to hyperoxidizing conditions within the ER, however, will inactivate Ero1p which acts as a catalyst for Pdi1p, and thus can lead to an increased presence of reduced thiols in protein domains (such as the EF-SAM domain of STIM) located in the ER lumen (Sevier et al. 2007). "
ABSTRACT: Abstract Store-operated Ca(2+) entry (SOCE) is a widespread mechanism in cells to raise cytosolic Ca(2+) and to refill Ca(2+) stores. T cells critically rely on SOCE mediated by stromal interaction molecules (STIM) and Orai molecules for their activation and regulation of gene transcription; cells such as muscle cells, neurons or melanocytes probably utilize SOCE for the transmission of inducible receptor-mediated function as well as for generalized Ca(2+) homeostasis mechanisms. Exposure to environmental or cell-intrinisic reactive oxygen species (ROS) can affect several components involved in Ca(2+) homeostasis and thus alter multiple pathways. While all cells have a capacity to produce intracellular ROS, exposure of immune and skin cells to extracellular oxidative stress is particularly high during inflammation and/or with UV exposure. This review briefly summarizes cell-intrinsic sources of ROS and focuses on current findings and controversies regarding the regulation of STIM and Orai by oxidative modifications. We also introduce melanocytes as a new model system to study the function of STIM and Orai isoforms under physiological conditions that include exposure to UV light as an activating stimulus.The Journal of Physiology 05/2012; 590(Pt 17):4193-200. DOI:10.1113/jphysiol.2012.230565 · 4.54 Impact Factor
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- "The results also unveiled a Trx oxidative pathway that became active shortly after illumination with low to moderate light intensity. To identify the oxidizing partner of ACHT1, we took advantage of the methodology that trapped in vivo ERO1, the thiol oxidase of PDI, and PDI in intermolecular disulfide complexes (Pollard et al., 1998; Frand and Kaiser, 1999; Mezghrani et al., 2001). The trapping in planta of ACHT1 reaction intermediates, in which ACHT1 is linked to its redox partner by an intermolecular disulfide bridge, was important for maintaining physiological conditions. "
ABSTRACT: The transition from dark to light involves marked changes in the redox reactions of photosynthetic electron transport and in chloroplast stromal enzyme activity even under mild light and growth conditions. Thus, it is not surprising that redox regulation is used to dynamically adjust and coordinate the stromal and thylakoid compartments. While oxidation of regulatory proteins is necessary for the regulation, the identity and the mechanism of action of the oxidizing pathway are still unresolved. Here, we studied the oxidation of a thylakoid-associated atypical thioredoxin-type protein, ACHT1, in the Arabidopsis thaliana chloroplast. We found that after a brief period of net reduction in plants illuminated with moderate light intensity, a significant oxidation reaction of ACHT1 arises and counterbalances its reduction. Interestingly, ACHT1 oxidation is driven by 2-Cys peroxiredoxin (Prx), which in turn eliminates peroxides. The ACHT1 and 2-Cys Prx reaction characteristics in plants further indicated that ACHT1 oxidation is linked with changes in the photosynthetic production of peroxides. Our findings that plants with altered redox poise of the ACHT1 and 2-Cys Prx pathway show higher nonphotochemical quenching and lower photosynthetic electron transport infer a feedback regulatory role for this pathway.The Plant Cell 05/2012; 24(5):1894-906. DOI:10.1105/tpc.112.097139 · 9.58 Impact Factor
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- "If reduced Pdi1p were essential for activating Ero1p, de pletion of Pdi1p from the ER would be expected to decrease activation of Ero1p by reduction of regulatory bonds even under prevailing reducing conditions in the ER. We have established conditions to deplete a cell of Pdi1p, which is essential for via bility, by glucose repression for 15 h of P GAL1 -PDI1 in a pdi1 genetic background (Fig. 5 B; Tachibana and Stevens, 1992; Frand and Kaiser, 1999). As expected, Pdi1p depletion disrupts folding in the ER, as shown by the accumulation of the ER form of carboxypeptidase Y (CPY), similar to that in cells that had been treated with DTT (Fig. 5 C). "
ABSTRACT: 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.The Journal of Cell Biology 03/2012; 196(6):713-25. DOI:10.1083/jcb.201110090 · 9.69 Impact Factor