scholarly journals A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

2019 ◽  
Author(s):  
Leticia Lemus ◽  
Zrinka Matić ◽  
Veit Goder
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Proteolytic Activity ◽  
Secretory Pathway ◽  
Model Organism ◽  
Degradation Pathway ◽  
Vacuolar Membrane ◽  
List Type ◽  
Er Quality Control

SummaryNewly synthesized proteins of the secretory pathway are quality-controlled inside the endoplasmic reticulum (ER) and, if not properly folded, are retained. An exception are glycosylphosphatidylinositol-anchored proteins (GPI-APs) which can leave the ER even when misfolded and are routed to the vacuole/lysosome for degradation by largely unknown mechanisms linked to post-ER quality control. Using yeast as model organism, we show that Gas1*, an ER-exported misfolded GPI-AP, is diverted from the secretory pathway to endosomes for transport to the vacuole. However, Gas1* is not sorted into endosomal intraluminal vesicles but internalizes directly from the vacuolar membrane. There, the vacuolar protease Pep4, but not any other known vacuolar protease, is required for Gas1* internalization. Our data reveal novel and unexpected mechanisms for invaginations from the vacuolar membrane.HighlightsER-exited misfolded GPI-anchored proteins are routed to the vacuole via endosomes but do not internalize into intraluminal vesiclesInternalization occurs directly from the vacuolar membrane into intravacuolar mobile structuresInternalization from the vacuolar membrane depends on the proteolytic activity of the vacuolar protease Pep4

scholarly journals A Striking Quality Control Subcompartment in Saccharomyces cerevisiae: The Endoplasmic Reticulum-associated Compartment

2004 ◽  
Vol 15 (2) ◽  
pp. 908-921 ◽  
Author(s):  
Gregory Huyer ◽  
Gaby L. Longsworth ◽  
Deborah L. Mason ◽  
Monica P. Mallampalli ◽  
J. Michael McCaffery ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Cellular Functions ◽  
Er Quality Control ◽  
Fluorescence And Electron Microscopy ◽  
The Unfolded Protein Response ◽  
Mutant Forms

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.

scholarly journals Chaperone-Mediated Reflux of Secretory Proteins to the Cytosol During Endoplasmic Reticulum Stress

2019 ◽  
Author(s):  
Aeid Igbaria ◽  
Philip I. Merksamer ◽  
Ala Trusina ◽  
Firehiwot Tilahun ◽  
Jefferey R. Johnson ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Control Process ◽  
Secretory Proteins ◽  
Er Quality Control ◽  
Quality Control Process

ABSTRACTDiverse 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 agents 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 S. cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and co-chaperones 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.SIGNIFICANCEApproximately one third of eukaryotic proteins are synthesized on ribosomes attached to the endoplasmic reticulum (ER) membrane. Many of these polypeptides co- or post-translationally translocate into the ER, wherein they fold and mature. An ER quality-control system proofreads these proteins by facilitating their folding and modification, while eliminating misfolded proteins through ER-associated degradation (ERAD). Yet, the fate of many secretory proteins during ER stress is not completely understood. Here, we uncovered an ER-stress induced “protein reflux” system that delivers intact, folded ER luminal proteins back to the cytosol without degrading them. We found that ER protein reflux works in parallel to ERAD and requires distinct ER-resident and cytosolic chaperones and co-chaperones.

scholarly journals The degradation pathway of a model misfolded protein is determined by aggregation propensity

2018 ◽  
Vol 29 (12) ◽  
pp. 1422-1434 ◽  
Author(s):  
Zhihao Sun ◽  
Jeffrey L. Brodsky
Keyword(s):  
Quality Control ◽  
Secretory Pathway ◽  
Multivesicular Body ◽  
Degradation Pathway ◽  
Misfolded Proteins ◽  
Protein Homeostasis ◽  
Substrate Selection ◽  
Er Quality Control ◽  
Aggregation Propensity ◽  
Novel Model

Protein homeostasis in the secretory pathway is maintained by a hierarchy of quality control checkpoints, including endoplasmic reticulum–associated degradation (ERAD), which leads to the destruction of misfolded proteins in the ER, as well as post-ER proteolysis. Although most aberrant proteins are degraded by ERAD, some misfolded proteins escape the ER and are degraded instead by lysosomal/vacuolar proteases. To date, it remains unclear how misfolded membrane proteins are selected for these different fates. Here we designed a novel model substrate, SZ*, to investigate how substrate selection is mediated in yeast. We discovered that SZ* is degraded by both the proteasome and vacuolar proteases, the latter of which occurs after ER exit and requires the multivesicular body pathway. By interrogating how various conditions affect the fate of SZ*, we also discovered that heat-shock and substrate overexpression increase ERAD targeting. These conditions also increase substrate aggregation. We next found that aggregation of the membrane-free misfolded domain in SZ* is concentration dependent, and fusion of this misfolded domain to a post-ER quality control substrate instead targets the substrate for ERAD. Our data indicate that a misfolded membrane protein with a higher aggregation propensity is preferentially retained in the ER and targeted for ERAD.

scholarly journals Inhibiting Endoplasmic Reticulum (ER)-associated Degradation of Misfolded Yor1p Does Not Permit ER Export Despite the Presence of a Diacidic Sorting Signal

2007 ◽  
Vol 18 (9) ◽  
pp. 3398-3413 ◽  
Author(s):  
Silvere Pagant ◽  
Leslie Kung ◽  
Mariana Dorrington ◽  
Marcus C.S. Lee ◽  
Elizabeth A. Miller
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Er Quality Control ◽  
Er Retention ◽  
Copii Vesicles ◽  
Er Export ◽  
Cystic Fibrosis Transmembrane

Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the “B-site” cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-ΔF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-ΔF by inhibiting its degradation does not permit access of Yor1p-ΔF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.

scholarly journals Altered Quality Control in the Endoplasmic Reticulum Causes Cortical Dysplasia in Knock-In Mice Expressing a Mutant BiP

2007 ◽  
Vol 28 (1) ◽  
pp. 293-301 ◽  
Author(s):  
Naoya Mimura ◽  
Shigeki Yuasa ◽  
Miho Soma ◽  
Hisayo Jin ◽  
Keita Kimura ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Neurological Disorders ◽  
Secretory Pathway ◽  
Cortical Dysplasia ◽  
Layer Formation ◽  
Er Quality Control ◽  
Physiological Processes ◽  
Neurological Phenotype ◽  
Disordered Layer

ABSTRACT Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) molecular chaperone that is central to ER function. We examined knock-in mice expressing a mutant BiP in order to elucidate physiological processes that are sensitive to BiP functions during development and adulthood. The mutant BiP lacked the retrieval sequence that normally functions to return BiP to the ER from the secretory pathway. This allowed us to examine the effects of a defect in ER function without completely eliminating BiP function. The homozygous mutant BiP neonates died after birth due to respiratory failure. Besides that, the mutant BiP mice displayed disordered layer formation in the cerebral cortex and cerebellum, a neurological phenotype of reeler mutant-like malformation. Consistent with the phenotype, Cajal-Retzius (CR) cells did not secrete reelin, and the expression of reelin was markedly reduced posttranscriptionally. Furthermore, the reduction in the size of the whole brain and the apparent scattering of CR cells throughout the cortex, which were distinct from the reeler phenotype, were also seen. These findings suggest that the maturation and secretion of reelin in CR cells and other factors related to neural migration may be sensitive to aberrant ER quality control, which may cause various neurological disorders.

scholarly journals Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation

2005 ◽  
Vol 169 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Eric D. Spear ◽  
Davis T.W. Ng
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Protein ◽  
Carboxypeptidase Y ◽  
Er Quality Control ◽  
Systematic Analysis ◽  
Proteinase A ◽  
Context Specific ◽  
The Relationship ◽  
Erad Pathway

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed “ER quality control” prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

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 Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

2015 ◽  
Vol 26 (3) ◽  
pp. 390-405 ◽  
Author(s):  
Abla Tannous ◽  
Nishant Patel ◽  
Taku Tamura ◽  
Daniel N. Hebert
Keyword(s):  
Quality Control ◽  
Secretory Pathway ◽  
Selection Process ◽  
Control Process ◽  
Degradation Pathway ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Mutant Proteins ◽  
Quality Control Process ◽  
Glycoprotein Secretion

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.

scholarly journals Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum

2004 ◽  
Vol 15 (6) ◽  
pp. 2537-2548 ◽  
Author(s):  
Satomi Nadanaka ◽  
Hiderou Yoshida ◽  
Fumi Kano ◽  
Masayuki Murata ◽  
Kazutoshi Mori
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Control System ◽  
Golgi Apparatus ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
Protein Response

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.

scholarly journals Endoplasmic reticulum chaperones participate in human cytomegalovirus US2-mediated degradation of class I major histocompatibility complex molecules

2008 ◽  
Vol 89 (5) ◽  
pp. 1122-1130 ◽  
Author(s):  
Kristina Oresic ◽  
Domenico Tortorella
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Major Histocompatibility Complex ◽  
Human Cytomegalovirus ◽  
Class I ◽  
Cell Surface Expression ◽  
Major Histocompatibility ◽  
Er Quality Control ◽  
Heavy Chains ◽  
Histocompatibility Complex

Inhibition of cell-surface expression of major histocompatibility complex class I molecules by human cytomegalovirus (HCMV, a β-herpesvirus) promotes escape from recognition by CD8+ cytotoxic T cells. The HCMV US2 and US11 gene products induce class I downregulation during the early phase of HCMV infection by facilitating the degradation of class I heavy chains. The HCMV proteins promote the transport of the class I heavy chains across the endoplasmic reticulum (ER) membrane into the cytosol by a process referred to as ‘dislocation’, which is then followed by proteasome degradation. This process has striking similarities to the degradation of misfolded ER proteins mediated by ER quality control. Even though the major steps of the dislocation reaction have been characterized, the cellular proteins, specifically the ER chaperones involved in targeting class I for dislocation, have not been fully delineated. To elucidate the chaperones involved in HCMV-mediated class I dislocation, we utilized a chimeric class I heavy chain with an affinity tag at its carboxy terminus. Interestingly, US2 but not US11 continued to target the class I chimera for destruction, suggesting a structural limitation for US11-mediated degradation. Association studies in US2 cells and in cells that express a US2 mutant, US2–186HA, revealed that class I specifically interacts with calnexin, BiP and calreticulin. These findings demonstrate that US2-mediated class I destruction utilizes specific chaperones to facilitate class I dislocation. The data suggest a more general model in which the chaperones that mediate protein folding may also function during ER quality control to eliminate aberrant ER proteins.

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