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Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

Kakihana T, Araki K, Vavassori S, Iemura S, Cortini M, Fagioli C, Natsume T, Sitia R, Nagata K - J. Biol. Chem. (2013)

Bottom Line: Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion.PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4.The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8397, Japan.

ABSTRACT
In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

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Prx4 and Ero1α share similar partners and subcellular localizations.A, 24 h after transfection with pcDNA3.1, Prx4-FLAG, or Ero1α-FLAG, 106 HeLa cells were incubated with or without 0.25 μm dithiobis succinimidyl propionate (DSP) on ice. Anti-FLAG immunoprecipitates (IP) were then eluted by FLAG peptides and analyzed by Western blot with the indicated antibodies. Aliquots of the total Nonidet P-40 lysates from 104 cells (INPUT) were loaded to estimate (co)-immunoprecipitation efficiency. B, lysates from 107 mouse myeloma J558L cells or their derivative expressing nitrophenol-specific secretory Ig-μ chains (J[μs]) were immunoprecipitated with anti-ERp44 and analyzed by Western blot with the indicated antibodies. The slightly more abundant Prx4 associated to ERp44 in J[μs] cells may reflect physiological interactions in the presence of an abundant substrate (7). C, HeLa cells were fixed by 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Co-localization of Prx4 or Ero1α with PDI or ERp44 was observed by immunofluorescence using the indicated fluorochrome-conjugated antibodies, as described under “Experimental Procedures.” g, green. r, red.
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Figure 1: Prx4 and Ero1α share similar partners and subcellular localizations.A, 24 h after transfection with pcDNA3.1, Prx4-FLAG, or Ero1α-FLAG, 106 HeLa cells were incubated with or without 0.25 μm dithiobis succinimidyl propionate (DSP) on ice. Anti-FLAG immunoprecipitates (IP) were then eluted by FLAG peptides and analyzed by Western blot with the indicated antibodies. Aliquots of the total Nonidet P-40 lysates from 104 cells (INPUT) were loaded to estimate (co)-immunoprecipitation efficiency. B, lysates from 107 mouse myeloma J558L cells or their derivative expressing nitrophenol-specific secretory Ig-μ chains (J[μs]) were immunoprecipitated with anti-ERp44 and analyzed by Western blot with the indicated antibodies. The slightly more abundant Prx4 associated to ERp44 in J[μs] cells may reflect physiological interactions in the presence of an abundant substrate (7). C, HeLa cells were fixed by 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Co-localization of Prx4 or Ero1α with PDI or ERp44 was observed by immunofluorescence using the indicated fluorochrome-conjugated antibodies, as described under “Experimental Procedures.” g, green. r, red.

Mentions: The ER oxidases Ero1α and Prx4 have at least two common features; one is their function in oxidative protein folding, and the other is their lack of intrinsic ER retention signals. Surprisingly, the latter feature is 100% conserved among Ero1α orthologs and 94.4% conserved among Prx4 in vertebrates (KEGG database (25)) (supplemental Fig. 1). To identify proteins involved in its subcellular localization, we performed LC-MS/MS analyses of the material co-immunoprecipitated with FLAG-tagged Prx4 and identified ERp44, PDI, ERp72, ERp46, and P5 (supplemental Fig. 2) (see also Ref. 18), yielding a pattern similar to what is reported for Ero1α. To further compare the interactomes of the two enzymes and provide additional specificity controls, we overexpressed Prx4-FLAG or Ero1α-FLAG in HeLa cells and analyzed the immunoprecipitates obtained with or without prior cross-linking with dithiobis succinimidyl propionate. Western blot analyses of the material specifically eluted with FLAG peptides confirmed that both Prx4 and Ero1α interact with ERp44, PDI, ERp72, P5, and ERp46 (Fig. 1A). The similar binding patterns are in line with coordinated roles of Prx4 and Ero1α in oxidative protein folding (26).


Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

Kakihana T, Araki K, Vavassori S, Iemura S, Cortini M, Fagioli C, Natsume T, Sitia R, Nagata K - J. Biol. Chem. (2013)

Prx4 and Ero1α share similar partners and subcellular localizations.A, 24 h after transfection with pcDNA3.1, Prx4-FLAG, or Ero1α-FLAG, 106 HeLa cells were incubated with or without 0.25 μm dithiobis succinimidyl propionate (DSP) on ice. Anti-FLAG immunoprecipitates (IP) were then eluted by FLAG peptides and analyzed by Western blot with the indicated antibodies. Aliquots of the total Nonidet P-40 lysates from 104 cells (INPUT) were loaded to estimate (co)-immunoprecipitation efficiency. B, lysates from 107 mouse myeloma J558L cells or their derivative expressing nitrophenol-specific secretory Ig-μ chains (J[μs]) were immunoprecipitated with anti-ERp44 and analyzed by Western blot with the indicated antibodies. The slightly more abundant Prx4 associated to ERp44 in J[μs] cells may reflect physiological interactions in the presence of an abundant substrate (7). C, HeLa cells were fixed by 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Co-localization of Prx4 or Ero1α with PDI or ERp44 was observed by immunofluorescence using the indicated fluorochrome-conjugated antibodies, as described under “Experimental Procedures.” g, green. r, red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795256&req=5

Figure 1: Prx4 and Ero1α share similar partners and subcellular localizations.A, 24 h after transfection with pcDNA3.1, Prx4-FLAG, or Ero1α-FLAG, 106 HeLa cells were incubated with or without 0.25 μm dithiobis succinimidyl propionate (DSP) on ice. Anti-FLAG immunoprecipitates (IP) were then eluted by FLAG peptides and analyzed by Western blot with the indicated antibodies. Aliquots of the total Nonidet P-40 lysates from 104 cells (INPUT) were loaded to estimate (co)-immunoprecipitation efficiency. B, lysates from 107 mouse myeloma J558L cells or their derivative expressing nitrophenol-specific secretory Ig-μ chains (J[μs]) were immunoprecipitated with anti-ERp44 and analyzed by Western blot with the indicated antibodies. The slightly more abundant Prx4 associated to ERp44 in J[μs] cells may reflect physiological interactions in the presence of an abundant substrate (7). C, HeLa cells were fixed by 4% paraformaldehyde and permeabilized by 0.2% Triton X-100. Co-localization of Prx4 or Ero1α with PDI or ERp44 was observed by immunofluorescence using the indicated fluorochrome-conjugated antibodies, as described under “Experimental Procedures.” g, green. r, red.
Mentions: The ER oxidases Ero1α and Prx4 have at least two common features; one is their function in oxidative protein folding, and the other is their lack of intrinsic ER retention signals. Surprisingly, the latter feature is 100% conserved among Ero1α orthologs and 94.4% conserved among Prx4 in vertebrates (KEGG database (25)) (supplemental Fig. 1). To identify proteins involved in its subcellular localization, we performed LC-MS/MS analyses of the material co-immunoprecipitated with FLAG-tagged Prx4 and identified ERp44, PDI, ERp72, ERp46, and P5 (supplemental Fig. 2) (see also Ref. 18), yielding a pattern similar to what is reported for Ero1α. To further compare the interactomes of the two enzymes and provide additional specificity controls, we overexpressed Prx4-FLAG or Ero1α-FLAG in HeLa cells and analyzed the immunoprecipitates obtained with or without prior cross-linking with dithiobis succinimidyl propionate. Western blot analyses of the material specifically eluted with FLAG peptides confirmed that both Prx4 and Ero1α interact with ERp44, PDI, ERp72, P5, and ERp46 (Fig. 1A). The similar binding patterns are in line with coordinated roles of Prx4 and Ero1α in oxidative protein folding (26).

Bottom Line: Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion.PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4.The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8397, Japan.

ABSTRACT
In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

Show MeSH
Related in: MedlinePlus