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Title Forcible destruction of severely misfolded mammalianglycoproteins by the non-glycoprotein ERAD pathway.
Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki;Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro;Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi;Kamiya, Yukiko; Kato, Koichi; Mori, Kazutoshi
Citation The Journal of cell biology (2015), 211(4): 775-784
Issue Date 2015-11-16
2015 Ninagawa et al.; This article is distributed under theterms of an AttributionNoncommercialShare AlikeNoMirror Sites license for the first six months after the publicationdate (see http://www.rupress.org/terms). After six months it isavailable under a Creative Commons License(AttributionNoncommercialShare Alike 3.0 Unportedlicense, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
Type Journal Article
The Rockefeller University Press $30.00J. Cell Biol. Vol. 211 No. 4 775784www.jcb.org/cgi/doi/10.1083/jcb.201504109
Soluble or transmembrane proteins in the ER possessing un-folded or misfolded parts in their luminal regions are retrotrans-located back to the cytoplasm and degraded by the proteasome, a series of events termed ER-associated degradation (ERAD)-L (Xie and Ng, 2010; Smith et al., 2011; Brodsky, 2012).
ERAD-L deals with both glycoproteins (gpERAD) and non-glycoproteins (non-gpERAD). Misfolded parts of non-gly-coproteins are most likely recognized by molecular chaperones represented by BiP. In contrast, glycoprotein degradation relies on the structure of N-glycans present on the surface of ERAD-L substrates. Glc3Man9GlcNAc2 attached to the asparagine resi-due is trimmed by glucosidase I and II to Man9GlcNAc2, which is in turn trimmed to Man8GlcNAc2 and then to Man7GlcNAc2. Man7GlcNAc2 exposing 1,6-linked mannose is recognized by the lectin molecule for the delivery of ERAD-L substrates to
the HRD1 E3 complex present in the ER membrane (Molinari, 2007; Hosokawa et al., 2010; Kamiya et al., 2012).
We have recently conducted comprehensive gene knock-out (KO) analyses to precisely identify the roles of the four 1,2-mannosidase candidates for the mannose trimming that occurs during ERAD-L, namely ER mannosidase I (ERmanI), EDEM1, EDEM2, and EDEM3. These experiments were per-formed in chicken DT40 cells using conventional homologous recombination and in human HCT116 cells using the tran-scription activator-like effector nuclease method. Our results were surprising (Ninagawa et al., 2014). Initial trimming from Man9GlcNAc2 to Man8GlcNAc2 was conducted by EDEM2, which had been thought to have no mannosidase activity (Mast et al., 2005), whereas subsequent trimming from Man8Glc-NAc2 to Man7GlcNAc2 was performed mostly by EDEM3 and to a slight extent by EDEM1. ERmanI appeared to play only a minor role in gpERAD.
Here, by constructing and analyzing DT40 and HCT116 cells simultaneously deficient in EDEM1/2/3, we show that dif-ferent ERAD pathways are used depending on the severity of
Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trim-ming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6, ATF6(C), CD3-TM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycopro-teins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6(C), CD3-TM, and EMC1, was de-layed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.
Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway
SatoshiNinagawa,1,3,4 TetsuyaOkada,1 YoshikiSumitomo,1 SatoshiHorimoto,1 TakehiroSugimoto,1 TokiroIshikawa,1 ShunichiTakeda,2 TakashiYamamoto,5 TadashiSuzuki,6 YukikoKamiya,3,4 KoichiKato,3,4,7 and KazutoshiMori1
1Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan2Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan3Institute for Molecular Science and 4Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki 444-8787, Japan5Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan6Glycometabolome Team, Systems Glycobiology Research Group, RIK EN Global Research Cluster, Wako, Saitama 351-0198, Japan7Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya, 467-8603, Japan
2015 Ninagawa et al. This article is distributed under the terms of an AttributionNoncommercialShare AlikeNo Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org /terms). After six months it is available under a Creative Commons License (AttributionNoncommercialShare Alike 3.0 Unported license, as described at http ://creativecommons .org /licenses /by -nc -sa /3 .0 /).
Correspondence to Koichi Kato: email@example.com; or Kazutoshi Mori: firstname.lastname@example.orgY.Kamiyas present address is Graduate School of Engineering and EcoTopia Science Institute, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan.Abbreviations used in this paper: 1PI, 1-proteinase inhibitor; EndoH, endoglycosidase H; ERAD, ER-associated degradation; ERmanI, ER mannosidase I; KO, knockout; MEF, mouse embryonic fibroblast; NHK, null Hong Kong; PNGase, peptide :N -glycanase; PQQ, pyrroloquinoline quinone; TKO, triple KO; WT, wild type.
ber 23, 2015jcb.rupress.org
Published November 16, 2015
http://jcb-dataviewer.rupress.org/jcb/browse/10702Original image data can be found at: http://jcb.rupress.org/content/suppl/2015/11/04/jcb.201504109.DC1.html Supplemental Material can be found at:
This article has original data in the JCB Data Viewer
JCB Volume 211 NumBer 4 2015776
misfolding in the ERAD-L substrates and that non-gpERAD has a much more positive role in protein quality control in the ER than its previously proposed role as a backup for gpERAD under ER stress (Ushioda et al., 2013), as indicated by the finding that non-gpERAD is able to clear severely misfolded glycoproteins from the ER even in the absence of pharmacological ER stress.
Results and discussion
We constructed EDEM1/2/3 triple KO (TKO) in both DT40 (gEDEM-TKO; Fig. S1) and HCT116 (hEDEM-TKO; Fig. S2 A) cells, in which functional EDEM1, EDEM2, or EDEM3 mRNA was not expressed (Fig.1, A and C). gEDEM-TKO and hEDEM-TKO cells grew slightly more slowly than respective wild-type (WT) cells (Fig.1, A and C). However, ER stress was not elicited in these cells because the levels of three ER stress marker proteins, BiP, ATF4, and XBP1(S), were not elevated (Fig. S2 B). The N-glycan profiles of total cellular glycoproteins revealed that M9 level was markedly elevated in gEDEM-TKO and hEDEM-TKO cells, similarly to WT DT40 and HCT116 cells treated with kifunensine, an inhibitor of 1,2-mannosi-dases in the ER (Fig.1, B and D), as expected from our previ-ous results (Ninagawa et al., 2014). Accordingly, degradation of endogenous ATF6, an ERAD-Lm (m for membrane pro-tein) substrate containing three potential N-glycosylation sites (Fig.3A) was blocked almost completely in gEDEM-TKO and hEDEM-TKO cells (Fig.1, E and F). The extent of this block-ade was comparable with that observed in WT cells treated with MG132, a proteasome inhibitor, or kifunensine (Ninagawa et al., 2014). We also found that hATF6(C)-TAP, an artificial ERAD-Ls (s for soluble protein) substrate containing the lu-minal region of human ATF6 flanked by the N-terminal sig-nal sequence and C-terminal tandem affinity purification tag (Fig. 3 A), was stabilized in gEDEM-TKO cells (Fig. 1 G). During SDS-PAGE, hATF6(C)-TAP in gEDEM-TKO cells migrated slightly more slowly than in WT cells because of the defect in mannose trimming, and this difference in migration was lost after endoglycosidase H (EndoH) treatment (Fig.1H). Instability of the native protein ATF6 in WT cells may result from intrinsically disordered parts found in its luminal region (Rosenbaum et al., 2011).
We next examined the effect of EDEM-TKO on degra-dation of the null Hong Kong (NHK) variant of 1-proteinase inhibitor (1PI), the most frequently used ERAD-Ls substrate (Sifers et al., 1988). Similarly to the case of hATF6(C)-TAP, NHK in gEDEM-TKO cells migrated slightly more slowly than in WT cells during SDS-PAGE, and this difference in migration was lost after EndoH treatment (Fig.2A). In the case of NHK-QQQ, the nonglycosylated version of NHK and its migra