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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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Silencing ERp44 allows secretion of endogenous Prx4, but not Ero1α.A, secretion of endogenous Prx4 or Ero1α by HeLa cells was analyzed with RNAi for nonspecific (NS) ERp44 or PDI (lanes 1–5) or both (lane 6) by specific siRNAs. 72 h after transfection, cells were cultured in Opti-MEM for 6 h and analyzed as described in the legend for Fig. 2. B, immunofluorescence of HeLa cells transfected with nonspecific siRNA (siNS) or PDI siRNA (siPDI). Endogenous Prx4 or Ero1α was co-stained with endogenous ERp44. In PDI-silenced cells the co-localization of Prx4 or Ero1α with ERp44 was more intense, consistent with a backup role of ERp44. siERp44, ERp44 siRNA. C, strategy utilized to dissect the retention of Ero1α and Prx4.
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Figure 4: Silencing ERp44 allows secretion of endogenous Prx4, but not Ero1α.A, secretion of endogenous Prx4 or Ero1α by HeLa cells was analyzed with RNAi for nonspecific (NS) ERp44 or PDI (lanes 1–5) or both (lane 6) by specific siRNAs. 72 h after transfection, cells were cultured in Opti-MEM for 6 h and analyzed as described in the legend for Fig. 2. B, immunofluorescence of HeLa cells transfected with nonspecific siRNA (siNS) or PDI siRNA (siPDI). Endogenous Prx4 or Ero1α was co-stained with endogenous ERp44. In PDI-silenced cells the co-localization of Prx4 or Ero1α with ERp44 was more intense, consistent with a backup role of ERp44. siERp44, ERp44 siRNA. C, strategy utilized to dissect the retention of Ero1α and Prx4.

Mentions: In view of their different distributions along ESC (7, 8), PDI and ERp44 might exert sequential effects on the localization/retention of Ero1α and Prx4. Therefore, we compared the effects of silencing ERp44, PDI, or both on the secretion of endogenous Prx4 and Ero1α by HeLa cells (Fig. 4A). Individual siRNAs for ERp44 or PDI effectively silenced the respective targets (Fig. 4A, lanes 7–12, right panel). Lowering the levels of ERp44 greatly promoted secretion of endogenous Prx4 (Fig. 4A, lanes 1–3, upper), but only marginally affected Ero1α retention (Fig. 4A, lanes 1–3, lower, and Fig. 4C, upper). Thus, under physiological conditions, PDI seems to retain Ero1α sufficiently. Neither endogenous Prx4 nor Ero1α was released by lowering the levels of PDI alone in HeLa cells (Fig. 4A, lanes 4 and 5, and Fig. 4C, middle). Considering that ERp44 is localized downstream with respect to PDI in the ESC, we surmised that ERp44 acted as a backup retention machinery in the absence of PDI (Fig. 4C, middle). Accordingly, the simultaneous silencing of ERp44 and PDI allowed secretion of both endogenous Ero1α and endogenous Prx4 by HeLa cells (Fig. 4A, lane 6). Backup mechanism by ERp44 was further confirmed by immunofluorescence of HeLa cells transfected with nonspecific siRNA or specific PDI. Endogenous PDI was efficiently silenced by RNAi (supplemental Fig. 4). As expected, co-localization of ERp44 with Ero1α and Prx4 was increased in PDI-silenced cells (Fig. 5B), whereas such a condition did not affect the morphology of the ER or ERGIC (supplemental Fig. 4), suggesting that retention of Ero1α and Prx4 in ESC depends mostly on ERp44 in the absence of PDI. Thus, sequential interactions with PDI and ERp44 underlie the intracellular retention of Prx4 and Ero1α. Ero1α displays higher affinity for PDI, but in its absence, it can be retrieved by ERp44. On the other hand, Prx4 is mainly retained by ERp44 because of its lower affinity for PDI (Fig. 3A).


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)

Silencing ERp44 allows secretion of endogenous Prx4, but not Ero1α.A, secretion of endogenous Prx4 or Ero1α by HeLa cells was analyzed with RNAi for nonspecific (NS) ERp44 or PDI (lanes 1–5) or both (lane 6) by specific siRNAs. 72 h after transfection, cells were cultured in Opti-MEM for 6 h and analyzed as described in the legend for Fig. 2. B, immunofluorescence of HeLa cells transfected with nonspecific siRNA (siNS) or PDI siRNA (siPDI). Endogenous Prx4 or Ero1α was co-stained with endogenous ERp44. In PDI-silenced cells the co-localization of Prx4 or Ero1α with ERp44 was more intense, consistent with a backup role of ERp44. siERp44, ERp44 siRNA. C, strategy utilized to dissect the retention of Ero1α and Prx4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Silencing ERp44 allows secretion of endogenous Prx4, but not Ero1α.A, secretion of endogenous Prx4 or Ero1α by HeLa cells was analyzed with RNAi for nonspecific (NS) ERp44 or PDI (lanes 1–5) or both (lane 6) by specific siRNAs. 72 h after transfection, cells were cultured in Opti-MEM for 6 h and analyzed as described in the legend for Fig. 2. B, immunofluorescence of HeLa cells transfected with nonspecific siRNA (siNS) or PDI siRNA (siPDI). Endogenous Prx4 or Ero1α was co-stained with endogenous ERp44. In PDI-silenced cells the co-localization of Prx4 or Ero1α with ERp44 was more intense, consistent with a backup role of ERp44. siERp44, ERp44 siRNA. C, strategy utilized to dissect the retention of Ero1α and Prx4.
Mentions: In view of their different distributions along ESC (7, 8), PDI and ERp44 might exert sequential effects on the localization/retention of Ero1α and Prx4. Therefore, we compared the effects of silencing ERp44, PDI, or both on the secretion of endogenous Prx4 and Ero1α by HeLa cells (Fig. 4A). Individual siRNAs for ERp44 or PDI effectively silenced the respective targets (Fig. 4A, lanes 7–12, right panel). Lowering the levels of ERp44 greatly promoted secretion of endogenous Prx4 (Fig. 4A, lanes 1–3, upper), but only marginally affected Ero1α retention (Fig. 4A, lanes 1–3, lower, and Fig. 4C, upper). Thus, under physiological conditions, PDI seems to retain Ero1α sufficiently. Neither endogenous Prx4 nor Ero1α was released by lowering the levels of PDI alone in HeLa cells (Fig. 4A, lanes 4 and 5, and Fig. 4C, middle). Considering that ERp44 is localized downstream with respect to PDI in the ESC, we surmised that ERp44 acted as a backup retention machinery in the absence of PDI (Fig. 4C, middle). Accordingly, the simultaneous silencing of ERp44 and PDI allowed secretion of both endogenous Ero1α and endogenous Prx4 by HeLa cells (Fig. 4A, lane 6). Backup mechanism by ERp44 was further confirmed by immunofluorescence of HeLa cells transfected with nonspecific siRNA or specific PDI. Endogenous PDI was efficiently silenced by RNAi (supplemental Fig. 4). As expected, co-localization of ERp44 with Ero1α and Prx4 was increased in PDI-silenced cells (Fig. 5B), whereas such a condition did not affect the morphology of the ER or ERGIC (supplemental Fig. 4), suggesting that retention of Ero1α and Prx4 in ESC depends mostly on ERp44 in the absence of PDI. Thus, sequential interactions with PDI and ERp44 underlie the intracellular retention of Prx4 and Ero1α. Ero1α displays higher affinity for PDI, but in its absence, it can be retrieved by ERp44. On the other hand, Prx4 is mainly retained by ERp44 because of its lower affinity for PDI (Fig. 3A).

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