scholarly journals Htm1p-Pdi1p is a folding sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Misfolded Proteins ◽  
Licensing Factor ◽  
Folded Proteins

AbstractOur understanding of how the endoplasmic reticulum-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the Htm1p-Pdi1p complex acts as a folding-sensitive mannosidase for catalyzing this first committed step. We reconstitute this step in vitro with Htm1p-Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p-Pdi1p is a glycoprotein-specific mannosidase, which preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p-Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Vol 113 (28) ◽  
pp. E4015-E4024 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Disulfide Isomerase ◽  
Misfolded Proteins ◽  
Disulfide Isomerase ◽  
Licensing Factor ◽  
Folded Proteins ◽  
Protein Disulfide

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

2021 ◽  
Author(s):  
Ritaban Halder ◽  
Daniel A. Nissley ◽  
Ian Sitarik ◽  
Edward P. O’Brien
Keyword(s):  
Tertiary Structure ◽  
Molecular Level ◽  
Meta Analysis ◽  
Hydrophobic Surface ◽  
Misfolded Proteins ◽  
Similar Degree ◽  
Functional Protein ◽  
Folded Proteins ◽  
And Function

ABSTRACTSubpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone-bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli’s GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state – having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long-time scale changes in protein structure and function can persist in cells because some protein’s non-native states can bypass components of the proteostasis machinery.TEASERNear-native, misfolded protein conformations explain why some soluble proteins fail to refold in the presence of chaperones.

scholarly journals TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum

2005 ◽  
Vol 388 (2) ◽  
pp. 647-655 ◽  
Author(s):  
Gerco HASSINK ◽  
Marjolein KIKKERT ◽  
Sjaak van VOORDEN ◽  
Shiow-Ju LEE ◽  
Robbert SPAAPEN ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Ring Finger ◽  
Transmembrane Domains ◽  
Dependent Manner ◽  
N Terminus ◽  
Ubiquitin Conjugating Enzyme ◽  
New Gene

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

scholarly journals Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response

2005 ◽  
Vol 25 (3) ◽  
pp. 921-932 ◽  
Author(s):  
Jingshi Shen ◽  
Erik L. Snapp ◽  
Jennifer Lippincott-Schwartz ◽  
Ron Prywes
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Competitive Binding ◽  
Competition Model ◽  
Misfolded Proteins ◽  
Endoplasmic Reticulum Stress Response ◽  
Specific Substrate ◽  
Mutant Form ◽  
Dynamic Binding

ABSTRACT Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

scholarly journals EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

2020 ◽  
Vol 21 (10) ◽  
pp. 3468 ◽  
Author(s):  
Marioara Chiritoiu ◽  
Gabriela N. Chiritoiu ◽  
Cristian V. A. Munteanu ◽  
Florin Pastrama ◽  
N. Erwin Ivessa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Interaction ◽  
Proteasomal Degradation ◽  
Misfolded Proteins ◽  
Misfolded Protein ◽  
Protein Protein Interaction ◽  
Pathological Conditions ◽  
Er Homeostasis

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

scholarly journals Misfolded Proteins Traffic from the Endoplasmic Reticulum (ER) Due to ER Export Signals

2007 ◽  
Vol 18 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Margaret M. Kincaid ◽  
Antony A. Cooper
Keyword(s):  
Endoplasmic Reticulum ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Therapeutic Approaches ◽  
Important Distinction ◽  
Anterograde Transport ◽  
Er Retention ◽  
Folded Proteins ◽  
Er Export ◽  
Export Signal

Most misfolded secretory proteins remain in the endoplasmic reticulum (ER) and are degraded by ER-associated degradation (ERAD). However, some misfolded proteins exit the ER and traffic to the Golgi before degradation. Using model misfolded substrates, with or without defined ER exit signals, we found misfolded proteins can depart the ER by continuing to exhibit the functional export signals present in the corresponding correctly folded proteins. Anterograde transport of misfolded proteins utilizes the same machinery responsible for exporting correctly folded proteins. Passive ER retention, in which misfolded proteins fail to exit the ER due to the absence of exit signals or the inability to functionally present them, likely contributes to the retention of nonnative proteins in the ER. Intriguingly, compromising ERAD resulted in increased anterograde trafficking of a misfolded protein with an ER exit signal, suggesting that ERAD and ER exit machinery can compete for binding of misfolded proteins. Disabling ERAD did not result in transport of an ERAD substrate lacking an export signal. This is an important distinction for those seeking possible therapeutic approaches involving inactivating ERAD in anticipation of exporting a partially active protein.

scholarly journals In vitro FRET analysis of IRE1 and BiP association and dissociation upon endoplasmic reticulum stress

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Megan C Kopp ◽  
Piotr R Nowak ◽  
Natacha Larburu ◽  
Christopher J Adams ◽  
Maruf MU Ali
Keyword(s):  
Endoplasmic Reticulum ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Misfolded Proteins ◽  
Protein Substrates ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Substrate Binding Domain

The unfolded protein response (UPR) is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER). The primary step in UPR activation is the detection of misfolded proteins, the mechanism of which is unclear. We have previously suggested an allosteric mechanism for UPR induction (<xref ref-type="bibr" rid="bib3">Carrara et al., 2015</xref>) based on qualitative pull-down assays. Here, we develop an in vitro Förster resonance energy transfer (FRET) UPR induction assay that quantifies IRE1 luminal domain and BiP association and dissociation upon addition of misfolded proteins. Using this technique, we reassess our previous observations and extend mechanistic insight to cover other general ER misfolded protein substrates and their folded native state. Moreover, we evaluate the key BiP substrate-binding domain mutant V461F. The new experimental approach significantly enhances the evidence suggesting an allosteric model for UPR induction upon ER stress.

scholarly journals Regulation of retrotranslocation by p97-associated deubiquitinating enzyme ataxin-3

2006 ◽  
Vol 174 (7) ◽  
pp. 963-971 ◽  
Author(s):  
Qiuyan Wang ◽  
Lianyun Li ◽  
Yihong Ye
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Degradation ◽  
Spinocerebellar Ataxia ◽  
Misfolded Proteins ◽  
Deubiquitinating Enzyme ◽  
Present Evidence ◽  
Type 3 ◽  
Efficient Elimination ◽  
Er Membrane

Misfolded proteins of the endoplasmic reticulum undergo retrotranslocation to enter the cytosol where they are degraded by the proteasome. Retrotranslocation of many substrates requires an ATPase complex consisting of the p97 ATPase and a dimeric cofactor, Ufd1-Npl4. We report that efficient elimination of misfolded ER proteins also involves ataxin-3 (atx3), a p97-associated deubiquitinating enzyme mutated in type-3 spinocerebellar ataxia. Overexpression of an atx3 mutant defective in deubiquitination inhibits the degradation of misfolded ER proteins and triggers ER stress. Misfolded polypeptides stabilized by mutant atx3 are accumulated in part as polyubiquitinated form, suggesting an involvement of its deubiquitinating activity in ER-associated protein degradation regulation. We demonstrate that atx3 transiently associates with the ER membrane via p97 and the recently identified Derlin–VIMP complex, and its release from the membrane appears to be governed by both the p97 ATPase cycle and its own deubiquitinating activity. We present evidence that atx3 may promote p97-associated deubiquitination to facilitate the transfer of polypeptides from p97 to the proteasome.

scholarly journals O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation

2001 ◽  
Vol 12 (4) ◽  
pp. 1093-1101 ◽  
Author(s):  
Carol Harty ◽  
Sabine Strahl ◽  
Karin Römisch
Keyword(s):  
Endoplasmic Reticulum ◽  
Yeast Cells ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Wild Type ◽  
Folding Intermediates ◽  
Alpha Factor ◽  
Er Export ◽  
Sec61 Channel

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by proteinO-mannosyl transferase 2 (Pmt2p).O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion ofPMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.

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