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Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

Tsunoda S, Avezov E, Zyryanova A, Konno T, Mendes-Silva L, Pinho Melo E, Harding HP, Ron D - Elife (2014)

Bottom Line: Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins.ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin.Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Wellcome Trust MRC Institute of Metabolic Science, Cambridge, United Kingdom NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagged LDL-R and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 6) or after an additional chase period (indicated). The mobility of the ER and Golgi forms of the LDL-R on reducing (left) and non-reducing (right) SDS-PAGE is indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitation. (B) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘A’ above. Shown is a representative experiment reproduce three times with similar outcome. (C) Autoradiograph of an experiment similar in layout to that depicted in ‘A’ above. Where indicated, the cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘C’ above. Shown is a representative experiment reproduce three times with similar outcome. (E) Bar graph of total cellular glutathione in cells manipulated as in the experiment described in ‘C’ (shown is the mean ±SD, n = 3, *p<0.05). (F) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 expressed in the ER of mouse embryonic fibroblasts with genetic lesions compromising disulfide bond formation (Ero1am/m; Ero1bm/m; Prdx4m/y) following a brief reductive pulse with dithiothreitol followed by a washout. The cells co-expressed ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) marked at its C-terminus with an mCherry fluorescent probe. (G) Bar diagram of the half-time to recovery of oxidized roGFP2 following the reductive DTT pulse in ‘F’ above. Note that expression of active ChaC1 in the ER did not affect the rate of recovery of the sentinel disulfide in ER-localized roGFP2.DOI:http://dx.doi.org/10.7554/eLife.03421.008
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fig4: Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagged LDL-R and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 6) or after an additional chase period (indicated). The mobility of the ER and Golgi forms of the LDL-R on reducing (left) and non-reducing (right) SDS-PAGE is indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitation. (B) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘A’ above. Shown is a representative experiment reproduce three times with similar outcome. (C) Autoradiograph of an experiment similar in layout to that depicted in ‘A’ above. Where indicated, the cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘C’ above. Shown is a representative experiment reproduce three times with similar outcome. (E) Bar graph of total cellular glutathione in cells manipulated as in the experiment described in ‘C’ (shown is the mean ±SD, n = 3, *p<0.05). (F) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 expressed in the ER of mouse embryonic fibroblasts with genetic lesions compromising disulfide bond formation (Ero1am/m; Ero1bm/m; Prdx4m/y) following a brief reductive pulse with dithiothreitol followed by a washout. The cells co-expressed ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) marked at its C-terminus with an mCherry fluorescent probe. (G) Bar diagram of the half-time to recovery of oxidized roGFP2 following the reductive DTT pulse in ‘F’ above. Note that expression of active ChaC1 in the ER did not affect the rate of recovery of the sentinel disulfide in ER-localized roGFP2.DOI:http://dx.doi.org/10.7554/eLife.03421.008

Mentions: Conversion of the LDL-R from its ER to post-ER form was unaffected by the presence of either active or inactive form of ER-ChaC1 and proceeded with a half-life of ∼1 hr in presence or absence of an active glutathione-depleting enzyme in the ER (Figure 4A). The non-reducing gel revealed identical accelerated mobility of LDL-R at the earliest time point, regardless of expression of ChaC1, indicating that glutathione depletion also had no drastic effect on the earlier oxidative phase of LDL-R maturation (Figure 4A,B).10.7554/eLife.03421.008Figure 4.Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.


Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

Tsunoda S, Avezov E, Zyryanova A, Konno T, Mendes-Silva L, Pinho Melo E, Harding HP, Ron D - Elife (2014)

Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagged LDL-R and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 6) or after an additional chase period (indicated). The mobility of the ER and Golgi forms of the LDL-R on reducing (left) and non-reducing (right) SDS-PAGE is indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitation. (B) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘A’ above. Shown is a representative experiment reproduce three times with similar outcome. (C) Autoradiograph of an experiment similar in layout to that depicted in ‘A’ above. Where indicated, the cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘C’ above. Shown is a representative experiment reproduce three times with similar outcome. (E) Bar graph of total cellular glutathione in cells manipulated as in the experiment described in ‘C’ (shown is the mean ±SD, n = 3, *p<0.05). (F) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 expressed in the ER of mouse embryonic fibroblasts with genetic lesions compromising disulfide bond formation (Ero1am/m; Ero1bm/m; Prdx4m/y) following a brief reductive pulse with dithiothreitol followed by a washout. The cells co-expressed ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) marked at its C-terminus with an mCherry fluorescent probe. (G) Bar diagram of the half-time to recovery of oxidized roGFP2 following the reductive DTT pulse in ‘F’ above. Note that expression of active ChaC1 in the ER did not affect the rate of recovery of the sentinel disulfide in ER-localized roGFP2.DOI:http://dx.doi.org/10.7554/eLife.03421.008
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fig4: Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled LDL receptor (LDL-R) immunopurified from HeLa cells co-expressing the FLAG-tagged LDL-R and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 6) or after an additional chase period (indicated). The mobility of the ER and Golgi forms of the LDL-R on reducing (left) and non-reducing (right) SDS-PAGE is indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitation. (B) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘A’ above. Shown is a representative experiment reproduce three times with similar outcome. (C) Autoradiograph of an experiment similar in layout to that depicted in ‘A’ above. Where indicated, the cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D) Graphic presentation of the conversion of the ER to Golgi form of the LDL-R from ‘C’ above. Shown is a representative experiment reproduce three times with similar outcome. (E) Bar graph of total cellular glutathione in cells manipulated as in the experiment described in ‘C’ (shown is the mean ±SD, n = 3, *p<0.05). (F) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 expressed in the ER of mouse embryonic fibroblasts with genetic lesions compromising disulfide bond formation (Ero1am/m; Ero1bm/m; Prdx4m/y) following a brief reductive pulse with dithiothreitol followed by a washout. The cells co-expressed ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) marked at its C-terminus with an mCherry fluorescent probe. (G) Bar diagram of the half-time to recovery of oxidized roGFP2 following the reductive DTT pulse in ‘F’ above. Note that expression of active ChaC1 in the ER did not affect the rate of recovery of the sentinel disulfide in ER-localized roGFP2.DOI:http://dx.doi.org/10.7554/eLife.03421.008
Mentions: Conversion of the LDL-R from its ER to post-ER form was unaffected by the presence of either active or inactive form of ER-ChaC1 and proceeded with a half-life of ∼1 hr in presence or absence of an active glutathione-depleting enzyme in the ER (Figure 4A). The non-reducing gel revealed identical accelerated mobility of LDL-R at the earliest time point, regardless of expression of ChaC1, indicating that glutathione depletion also had no drastic effect on the earlier oxidative phase of LDL-R maturation (Figure 4A,B).10.7554/eLife.03421.008Figure 4.Maturation of the LDL-R receptor is unaffected by depletion of ER glutathione.

Bottom Line: Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins.ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin.Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Wellcome Trust MRC Institute of Metabolic Science, Cambridge, United Kingdom NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus