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 Limited ER quality control for GPI-anchored proteins

2016 ◽  
Vol 213 (6) ◽  
pp. 693-704 ◽  
Author(s):  
Natalia Sikorska ◽  
Leticia Lemus ◽  
Auxiliadora Aguilera-Romero ◽  
Javier Manzano-Lopez ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Misfolded Proteins ◽  
Control Mechanisms ◽  
Gpi Anchor ◽  
Er Quality Control ◽  
Entire Class ◽  
Er Export ◽  
Export Signal

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevant.

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.

scholarly journals Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress

2019 ◽  
Vol 116 (23) ◽  
pp. 11291-11298 ◽  
Author(s):  
Aeid Igbaria ◽  
Philip I. Merksamer ◽  
Ala Trusina ◽  
Firehiwot Tilahun ◽  
Jeffrey R. Johnson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Control Process ◽  
Secretory Proteins ◽  
Quality Control Process ◽  
Folded Proteins ◽  
Green Fluorescent

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

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.

Calnexin: a molecular chaperone with a taste for carbohydrate

1995 ◽  
Vol 73 (3-4) ◽  
pp. 123-132 ◽  
Author(s):  
David B. Williams
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Chaperone Function ◽  
Folded Proteins

Calnexin is an integral membrane protein of the endoplasmic reticulum (ER) that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged binding to misfolded or incompletely folded proteins. Recent studies have demonstrated that calnexin functions as a molecular chaperone to facilitate the folding and assembly of proteins in the ER. It is also a component of the quality control system that prevents proteins from progressing along the secretory pathway until they have acquired proper tertiary or quaternary structure. Most proteins that are translocated into the ER are glycosylated at Asn residues, and calnexin's interactions are almost exclusively restricted to proteins that possess this posttranslational modification. The preference for glycoproteins resides in calnexin's ability to function as a lectin with specificity for the GlC1Man9GlcNAc2 oligosaccharide, an early intermediate in the processing of Asn-linked oligosaccharides. Calnexin also has the capacity to bind to polypeptide segments of unfolded glycoproteins. Available evidence suggests that calnexin utilizes its lectin property during initial capture of a newly synthesized glycoprotein and that subsequent association (and chaperone function) is mediated through polypeptide interactions. Unlike other molecular chaperones that are soluble proteins, calnexin is an intrinsic component of the ER membrane. Its unique ability to capture unfolded glycoproteins through their large oligosaccharide moieties may have evolved as a means to overcome accessibility problems imposed by being constrained within a lipid bilayer.Key words: protein folding, molecular chaperones, calnexin, quality control, endoplasmic reticulum.

Protein Quality Control of the Endoplasmic Reticulum and Ubiquitin–Proteasome-Triggered Degradation of Aberrant Proteins: Yeast Pioneers the Path

2018 ◽  
Vol 87 (1) ◽  
pp. 751-782 ◽  
Author(s):  
Nicole Berner ◽  
Karl-Richard Reutter ◽  
Dieter H. Wolf
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Protein Quality ◽  
Protein Structures ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Structural Alterations ◽  
Ubiquitin Proteasome

Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin–proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.

scholarly journals BiP Availability Distinguishes States of Homeostasis and Stress in the Endoplasmic Reticulum of Living Cells

2010 ◽  
Vol 21 (12) ◽  
pp. 1909-1921 ◽  
Author(s):  
Chun Wei Lai ◽  
Deborah E. Aronson ◽  
Erik Lee Snapp
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Misfolding ◽  
Single Cells ◽  
Cell Types ◽  
Secretory Protein ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Molecular Events ◽  
The Unfolded Protein Response

Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.

scholarly journals LTK is an ER-resident receptor tyrosine kinase that regulates secretion

2019 ◽  
Author(s):  
Federica G. Centonze ◽  
Veronika Reiterer ◽  
Karsten Nalbach ◽  
Kota Saito ◽  
Krzysztof Pawlowski ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Receptor Tyrosine Kinase ◽  
Secretory Proteins ◽  
Golgi Transport ◽  
Er Exit Sites ◽  
Local Signaling ◽  
Er Export ◽  
Pharmacologic Inhibition

AbstractThe endoplasmic reticulum (ER) is a key regulator of cellular proteostasis because it controls folding, sorting and degradation of secretory proteins. Much has been learned about how environmentally triggered signaling pathways regulate ER function, but only little is known about local signaling at the ER. The identification of ER-resident signaling molecules will help gain a deeper understanding of the regulation of ER function and thus of proteostasis. Here, we show that leukocyte tyrosine kinase (LTK) is an ER-resident receptor tyrosine kinase. Depletion of LTK as well as its pharmacologic inhibition reduces the number of ER exit sites and slows ER-to-Golgi transport. Furthermore, we show that LTK interacts with and phosphorylates Sec12. Expression of a phosphoablating mutant of Sec12 reduces the efficiency of ER export. Thus, LTK-to-Sec12 signaling represents the first example of an ER-resident signaling module the potential to regulate proteostasis.

scholarly journals The proteasome biogenesis regulator Rpn4 cooperates with the unfolded protein response to promote resistance to endoplasmic reticulum stress

2018 ◽  
Author(s):  
Rolf M. Schmidt ◽  
Sebastian Schuck
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Protein Misfolding ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Multiple Signaling

ABSTRACTMisfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of RPN4 transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.

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