Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 May;2(5):415-22.
doi: 10.1093/embo-reports/kve084.

A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation

N Hosokawa  1 , I Wada, K Hasegawa, T Yorihuzi, L O Tremblay, A Herscovics, K Nagata
Affiliations

A novel ER alpha-mannosidase-like protein accelerates ER-associated degradation

N Hosokawa et al. EMBO Rep. 2001 May.

Abstract

The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER-associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing alpha-mannosidase-like protein (EDEM) is similar to class I alpha1,2-mannosidases involved in N-glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded alpha1-antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N-glycan dependent manner.

PubMed Disclaimer

Figures

None
Fig. 1. Peptide sequence of mouse EDEM and induction of Edem mRNA by ER stress. (A) Alignment of peptide sequences of mouse EDEM and human ER α1,2-mannosidase I (ER Man I). Identical residues are boxed. Two Cys residues conserved among processing α-mannosidases are shown by *, and Cys of EDEM are marked by open triangles. Conserved acidic amino acids are shown by arrowheads. Putative transmembrane regions are underlined, and possible N-glycosylation sites of mouse EDEM are dotted. (B) Northern blot analysis of mouse BALB/c 3T3 cells treated with various agents to induce ER stress (tunicamycin, 2-deoxyglucose, A23187) or cytoplasmic stress (heat shock, arsenite). Ten micrograms of total cellular RNA were hybridized with 32P-labelled cDNAs encoding EDEM, GRP78 (BiP), calnexin (CNX), β-actin, or Hsp70. (C) Northern blot analysis of various primate cell lines. Ten micrograms of total cellular RNA from control cells (C) or cells treated with tunicamycin for 4 h (Tu) were hybridized with 32P-labelled cDNAs encoding human homologue of EDEM (KIAA0212), ER Man I, GRP78 or CNX.
None
Fig. 2. EDEM-HA localizes in the ER. (A) Autoradiogram of in vitro translated EDEM-HA in rabbit reticulocyte lysate metabolically labelled with [35S]methionine. In vitro transcribed Edem-HA RNA was used as template. EDEM-HA became resistant to proteinase K digestion when it was translocated into the microsomes. An arrowhead indicates the position of N-glycosylated EDEM-HA within the microsomes (78 kDa), and an arrow shows EDEM-HA without N-glycosylation (69 kDa). Open triangle shows the position of band detected without RNA template, and filled circles denote the degradation or immature products which were detected by α-HA antibody. Molecular mass standards (kDa) are shown on the left. (B) Autoradiogram of immunoprecipitation from metabolically labelled COS-7 cells. Arrowheads indicate the transfected EDEM-HA, and open triangles show the 150 kDa associated cellular protein that decreases during the chase period. P, pulse label for 45 min; C, chase for 45 min.
None
Fig. 3. EDEM accelerates the degradation of misfolded A1AT glycoprotein. (A) Autoradiogram following SDS–PAGE of immunoprecipitated material from HEK 293 cells cotransfected with NHK and EDEM-HA, or transfected with NHK and mock (indicated by –). The position of the predicted Man9 form of the A1AT variant (NHK) is shown by *, and that of the Man8 form by **. Cells were pulse-labelled for 15 min with [35S]methionine, and chased for the periods indicated. In this experiment only, lysates of cells cotransfected with EDEM-HA containing 1.6-fold more TCA-insoluble radioactivity than mock-transfected cells were used, since if equal amounts were used, the intensity of NHK signals in cells cotransfected with EDEM-HA was reduced to 42 ± 5% of the signal in mock-transfected cells at 0 h chase [see (C)]. The relative radioactivity in NHK at different times of chase was plotted on a semi-log scale relative to the intensity observed at 0 h chase. Error bars indicate standard deviations from the average of four (mock) or six (EDEM-HA) independent experiments. (B) Autoradiogram showing coimmunoprecipitation of NHK and EDEM-HA. The gel was exposed heavily to show coimmunoprecipitated bands. An arrowhead indicates the position of NHK, arrows show the transfected EDEM-HA, and the open triangle denotes the 150 kDa associated protein. The non-specific band detected in the immunoprecipitates using protein G-Sepharose is shown by dots (··). EDEM-HA appears as a doublet of different glycosylated forms, since a single band was formed after PNGase F treatment (data not shown), and EDEM has five possible N-glycosylation sites (Figure 1A). Molecular mass standards (kDa) are shown on the left. (C) Inhibition of NHK degradation by proteasome inhibitors. Cells were pulse-labelled for 15 min (P) and chased for 90 min (C), in the presence or absence of the proteasome inhibitors, lactacystin or MG132. MG135, a calpain inhibitor, was used as a control of MG132 treatment (indicated by –). Signals from immunoprecipitated NHK were compared with those of mock-transfected cells without proteasome inhibitor during pulse-labelling. (D) Autoradiogram showing coimmunoprecipitation of NHK and EDEM-HA in cells treated with lactacystin. An arrowhead, arrows, open triangle and dots indicate the same species as in Figure 3B. Open circle denotes another coimmunoprecipitated bands. The right panel (lanes 9–16) shows a 2.5 times longer exposure than the gel shown in the left panel (lanes 1–8).
None
Fig. 4. EDEM accelerates the degradation of misfolded protein in an α1,2-mannosidase-dependent manner. (A) Autoradiogram showing the inhibition of NHK degradation by an α1,2-mannosidase inhibitor kifunensine. Metabolic labelling and immunoprecipitation of transfected HEK 293 cells was performed as described in Figure 3. The position of the predicted Man9 form of the A1AT NHK is shown by *, and that of the Man8 form by **. Intensity of NHK at 0 h chase without kifunensine was arbitrarily set to 1.0, and plotted as in Figure 3A. (B) Degradation of non-glycosylated protein in the ER. pEYFP-ER (Clontech, Palo Alto, CA) was transfected to HEK 293 cells and the intracellular fate of expressed GFP was examined with or without the coexpression of EDEM-HA, as described in A1AT NHK.
See this image and copyright information in PMC

References

    1. Bonifacino J.S. and Weissman, A.M. (1998) Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell. Dev. Biol., 14, 19–57. - PMC - PubMed
    1. Casagrande R., Stern, P., Diehn, M., Shamu, C., Osario, M., Zúñiga, M., Brown, P.O. and Ploegh, H. (2000) Degradation of proteins from the ER of S. cerevisiae requires an intact unfolded protein response pathway. Mol. Cell, 5, 729–735. - PubMed
    1. Chapman R., Sidrauski, C. and Walter, P. (1998) Intracellular signaling from the endoplasmic reticulum to the nucleus. Annu. Rev. Cell. Dev. Biol., 14, 459–485. - PubMed
    1. Crystal R.G. (1989) The α1-antitrypsin gene and its deficiency states. Trends Genet., 5, 411–417. - PubMed
    1. Ellgaard L., Molinari, M. and Helenius, A. (1999) Setting the standards: quality control in the secretory pathway. Science, 286, 1882–1888. - PubMed

Publication types

MeSH terms

Associated data