scholarly journals Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

2019 ◽  
Author(s):  
Julia D. Knopf ◽  
Nina Landscheidt ◽  
Cassandra L. Pegg ◽  
Benjamin L. Schulz ◽  
Nathalie Kühnle ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Quantitative Proteomics ◽  
Proteasomal Degradation ◽  
Rhomboid Protease ◽  
Protein Fragments ◽  
Clearance Mechanism ◽  
Endogenous Substrate ◽  
Erad Pathway ◽  
Substrate Spectrum

AbstractThe Endoplasmic Reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosacharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalyzed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. Thereby, RHBDL4 controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.

scholarly journals Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I

2003 ◽  
Vol 376 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Claudia KITZMÜLLER ◽  
Andrea CAPRINI ◽  
Stuart E. H. MOORE ◽  
Jean-Pierre FRÉNOY ◽  
Eva SCHWAIGER ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Sequence Of Events ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Degradation Intermediates ◽  
Erad Pathway

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI332, which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive α1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI332 in the ERAD pathway. In a first step, the Man8 isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI332 intermediates. Some other trimmed N-glycan species, in particular Glc1Man7GlcNAc2, were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI332 accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

scholarly journals RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets

2021 ◽  
Author(s):  
Viorica Liebe Lastun ◽  
Matthew Freeman
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Er Stress ◽  
Liver Steatosis ◽  
Physiological Role ◽  
Cell Types ◽  
Rhomboid Protease ◽  
Rapid Changes

In metazoans, the architecture of the endoplasmic reticulum (ER) differs between cell types, and undergoes major changes through the cell cycle and according to physiological needs. Although much is known about how the different ER morphologies are generated and maintained, especially the ER tubules, how context dependent changes in ER shape and distribution are regulated and the factors involved are less characterized. Here, we show that RHBDL4, an ER-resident rhomboid protease, modulates the shape and distribution of the ER, especially under conditions that require rapid changes in the ER sheet distribution, including ER stress. RHBDL4 interacts with CLIMP-63, a protein involved in ER sheet stabilisation, and with the cytoskeleton. Mice lacking RHBDL4 are sensitive to ER stress and develop liver steatosis, a phenotype associated with unresolved ER stress. Our data introduce a new physiological role of RHBDL4 and also imply that this function does not require its enzymatic activity.

scholarly journals ΔF508 CFTR Pool in the Endoplasmic Reticulum Is Increased by Calnexin Overexpression

2004 ◽  
Vol 15 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Tsukasa Okiyoneda ◽  
Kazutsune Harada ◽  
Motohiro Takeya ◽  
Kaori Yamahira ◽  
Ikuo Wada ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endoplasmic Reticulum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Cystic Fibrosis Transmembrane ◽  
Erad Pathway

The most common cystic fibrosis transmembrane conductance regulator (CFTR) mutant in cystic fibrosis patients, ΔF508 CFTR, is retained in the endoplasmic reticulum (ER) and is consequently degraded by the ubiquitin-proteasome pathway known as ER-associated degradation (ERAD). Because the prolonged interaction of ΔF508 CFTR with calnexin, an ER chaperone, results in the ERAD of ΔF508 CFTR, calnexin seems to lead it to the ERAD pathway. However, the role of calnexin in the ERAD is controversial. In this study, we found that calnexin overexpression partially attenuated the ERAD of ΔF508 CFTR. We observed the formation of concentric membranous bodies in the ER upon calnexin overexpression and that the ΔF508 CFTR but not the wild-type CFTR was retained in the concentric membranous bodies. Furthermore, we observed that calnexin overexpression moderately inhibited the formation of aggresomes accumulating the ubiquitinated ΔF508 CFTR. These findings suggest that the overexpression of calnexin may be able to create a pool of ΔF508 CFTR in the ER.

scholarly journals Yos9p assists in the degradation of certain nonglycosylated proteins from the endoplasmic reticulum

2011 ◽  
Vol 22 (16) ◽  
pp. 2937-2945 ◽  
Author(s):  
Laura A. Jaenicke ◽  
Holger Brendebach ◽  
Matthias Selbach ◽  
Christian Hirsch
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Quantitative Proteomics ◽  
Structural Defects ◽  
Sterol Synthesis ◽  
Mutant Variant ◽  
Substrate Spectrum

The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.

scholarly journals The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation

2020 ◽  
Vol 19 (11) ◽  
pp. 1896-1909
Author(s):  
Teresa M. Buck ◽  
Xuemei Zeng ◽  
Pamela S. Cantrell ◽  
Richard T. Cattley ◽  
Zikri Hasanbasri ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Proteasome System ◽  
Sterol Synthesis ◽  
High Confidence ◽  
Assembly Pathway ◽  
Affinity Isolation ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
Erad Pathway

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin–proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques—including MS—have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

scholarly journals Intramembrane protease RHBDL4 interacts with erlin complex to target unstable soluble proteins for degradation

2019 ◽  
Author(s):  
Nathalie Kühnle ◽  
Josephine Bock ◽  
Julia D. Knopf ◽  
Nina Landscheidt ◽  
Jin-Gu Lee ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Major Histocompatibility Complex ◽  
Protein Degradation ◽  
Major Histocompatibility ◽  
Lysosomal Degradation ◽  
Rhomboid Protease ◽  
Unfolded Protein ◽  
Histocompatibility Complex ◽  
Protein Response ◽  
Erad Pathway

AbstractProtein degradation is fundamentally important to ensure cell homeostasis. In the Endoplasmic Reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins into the cytoplasm for turnover by the proteasome. In contrast, lysosomal degradation serves as failsafe mechanism for removal of proteins that resist ERAD by forming aggregates. In previous work, we showed that the ER-resident rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear and its full substrate spectrum remains to be defined. Here, we show that besides membrane proteins, RHBDL4 cleaves aggregation-prone, luminal ERAD substrates including a soluble version of the major histocompatibility complex heavy chain (MHC202). RHBDL4’s interaction with erlin ERAD substrate receptors and reciprocal interaction of MHC202 with erlins suggest that RHBDL4 defines a substrate clipping mechanism that rescues aggregation-prone peptides in the ER lumen from terminal aggregation.AbbreviationsER, endoplasmic reticulum; ERAD, ER-associated degradation; MHC, major histocompatibility complex; TM, transmembrane; UPR, unfolded protein response.

scholarly journals Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Antonio Galeone ◽  
Joshua M Adams ◽  
Shinya Matsuda ◽  
Maximiliano F Presa ◽  
Ashutosh Pandey ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Secretory Pathway ◽  
Proteasomal Degradation ◽  
Bmp Signaling ◽  
Tissue Specific ◽  
Biologically Relevant ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Specific Manner

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

scholarly journals A deubiquitinase negatively regulates retro-translocation of nonubiquitinated substrates

2013 ◽  
Vol 24 (22) ◽  
pp. 3545-3556 ◽  
Author(s):  
Kaleena M. Bernardi ◽  
Jeffrey M. Williams ◽  
Takamasa Inoue ◽  
Aric Schultz ◽  
Billy Tsai
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Proteasomal Degradation ◽  
Binding Studies ◽  
Negative Regulators ◽  
Negative Function ◽  
Er Targeting ◽  
Membrane Bound

Endoplasmic reticulum (ER) membrane–bound E3 ubiquitin ligases promote ER-associated degradation (ERAD) by ubiquitinating a retro-translocated substrate that reaches the cytosol from the ER, targeting it to the proteasome for destruction. Recent findings implicate ERAD-associated deubiquitinases (DUBs) as positive and negative regulators during ERAD, reflecting the different consequences of deubiquitinating a substrate prior to proteasomal degradation. These observations raise the question of whether a DUB can control the fate of a nonubiquitinated ERAD substrate. In this study, we probed the role of the ERAD-associated DUB, YOD1, during retro-translocation of the nonubiquitinated cholera toxin A1 (CTA1) peptide, a critical intoxication step. Through combining knockdown, overexpression, and binding studies, we demonstrated that YOD1 negatively controls CTA1 retro-translocation, likely by deubiquitinating and inactivating ubiquitinated ERAD components that normally promote toxin retro-translocation. YOD1 also antagonizes the proteasomal degradation of nonglycosylated pro-α factor, a postulated nonubiquitinated yeast ERAD substrate, in mammalian cells. Our findings reveal that a cytosolic DUB exerts a negative function during retro-translocation of nonubiquitinated substrates, potentially by acting on elements of the ERAD machinery.

Localization of Intracisternal a Particle Antigens in Neoplastic Cells and Preimplantation Mouse Embryos

1980 ◽  
Vol 38 ◽  
pp. 696-697
Author(s):  
Thomas T.F. Huang ◽  
Patricia G. Calarco
Keyword(s):  
Endoplasmic Reticulum ◽  
Normal Development ◽  
Mouse Embryos ◽  
Neoplastic Cells ◽  
Large Numbers ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Intracisternal A Particle ◽  
Normal Feature

The stage specific appearance of a retravirus, termed the Intracisternal A particle (IAP) is a normal feature of early preimplantation development. To date, all feral and laboratory strains of Mus musculus and even Asian species such as Mus cervicolor and Mus pahari express the particles during the 2-8 cell stages. IAP form by budding into the endoplasmic reticulum and appear singly or as groups of donut-shaped particles within the cisternae (fig. 1). IAP are also produced in large numbers in several neoplastic cells such as certain plasmacytomas and rhabdomyosarcomas. The role of IAP, either in normal development or in neoplastic behavior, is unknown.

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