Golgi-mediated vacuolar sorting of the endoplasmic reticulum chaperone BiP may play an active role in quality control within the secretory pathway

Quality control in the endoplasmic reticulum (ER) prevents the arrival of incorrectly or incompletely folded proteins at their final destinations and targets permanently misfolded proteins for degradation. Such proteins have a high affinity for the ER chaperone BiP and are finally degraded via retrograde translocation from the ER lumen back to the cytosol. This ER-associated protein degradation (ERAD) is currently thought to constitute the main disposal route, but there is growing evidence for a vacuolar role in quality control. We show that BiP is transported to the vacuole in a wortmannin-sensitive manner in tobacco (Nicotiana tabacum) and that it could play an active role in this second disposal route. ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus, where it competes with other HDEL receptor ligands. When HDEL-mediated retrieval from the Golgi fails, BiP is transported to the lytic vacuole via multivesicular bodies, which represent the plant prevacuolar compartment. We also demonstrate that a subset of BiP-ligand complexes is destined to the vacuole and differs from those likely to be disposed of via the ERAD pathway. Vacuolar disposal could act in addition to ERAD to maximize the efficiency of quality control in the secretory pathway.     

Endoplasmic reticulum degradation. Reverse protein transport and its end in the proteasome

Degradation of misfolded or unassembled proteins of the secretory pathway is an essential function of the quality control system of the Endoplasmic Reticulum (ER). Using yeast as a model organism we show that a mutated and therefore misfolded soluble lumenal protein carboxypeptidase yscY (CPY*), and a polytopic membrane protein, the ATP-binding cassette transporter Pdr5 (Pdr5*), are retrograde transported out of the ER and degraded via the cytoplasmic ubiquitin-proteasome system. Retrograde transport depends on an intact Sec61 translocon. Complete import of CPY* into the lumen of the ER requests a new targeting mechanism for retrograde transport of the malfolded enzyme through the Sec61 channel to occur. For soluble CPY*, but not for the polytopic membrane protein Pdr5* action of the ER-lumenal Hsp70 chaperone Kar2 is necessary to deliver the protein to the ubiquitin-proteasome machinery. Polyubiquitination of CPY* and Pdr5* by the ubiquitin conjugating enzymes Ubc6 and Ubc7 is crucial for degradation to occur. Also transport of CPY* out of the ER-lumen depends on ubiquitination. Newly discovered proteins of the ER membrane, Der1, Der3/Hrd1, and Hrd3 are specifically involved in the retrograde transport processes.     

Degradation of endoplasmic reticulum (ER) quality control substrates requires transport between the ER and Golgi

Endoplasmic reticulum (ER) quality control (ERQC) components retain and degrade misfolded proteins, and our results have found that the degradation of the soluble ERQC substrates CPY* and PrA* but not membrane spanning ERQC substrates requires transport between the ER and Golgi. Stabilization of these misfolded soluble proteins was seen in cells lacking Erv29p, a probable Golgi localized protein that cycles through the ER by means of a di-lysine ER retrieval motif (KKKIY). Cells lacking Erv29p also displayed severely retarded ER exit kinetics for a subset of correctly folded proteins. We suggest that Erv29p is likely involved in cargo loading of a subset of proteins, including soluble misfolded proteins, into vesicles for ER exit. The stabilization of soluble ERQC substrates in both erv29Delta cells and sec mutants blocked in either ER exit (sec12) or vesicle delivery to the Golgi (sec18) suggests that ER-Golgi transport is required for ERQC and reveals a new aspect of the degradative mechanism.     

Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control

Misfolded proteins retained in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation pathway. The mechanisms used to sort them from correctly folded proteins remain unclear. Analysis of substrates with defined folded and misfolded domains has revealed a system of sequential checkpoints that recognize topologically distinct domains of polypeptides. The first checkpoint examines the cytoplasmic domains of membrane proteins. If a lesion is detected, it is retained statically in the ER and rapidly degraded without regard to the state of its other domains. Proteins passing this test face a second checkpoint that monitors domains localized in the ER lumen. Proteins detected by this pathway are sorted from folded proteins and degraded by a quality control mechanism that requires ER-to-Golgi transport. Although the first checkpoint is obligatorily directed at membrane proteins, the second monitors both soluble and membrane proteins. Our data support a model whereby "properly folded" proteins are defined biologically as survivors that endure a series of distinct checkpoints.     

Derlin-dependent retrograde transport from endosomes to the Golgi apparatus

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.     

Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum

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.     

Search and destroy: ER quality control and ER-associated protein degradation

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms.The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

Search and Destroy: ER Quality Control and ER-Associated Protein Degradation

Abstract

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

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

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.     

Inositol deacylation by Bst1p is required for the quality control of glycosylphosphatidylinositol-anchored proteins

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the beta-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins.     

Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

ER stress and its functional link to mitochondria: role in cell survival and death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.     

Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals

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.     

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.     

Selective targeting of proteins within secretory pathway for endoplasmic reticulum-associated degradation

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.     

A Highlights from MBoC Selection: Evasion of Endoplasmic Reticulum Surveillance Makes Wsc1p an Obligate Substrate of Golgi Quality Control

In the endoplasmic reticulum (ER), most newly synthesized proteins are retained by quality control mechanisms until folded. Misfolded molecules are sorted to ER-associated degradation (ERAD) pathways for disposal. Reports of mutant proteins degraded in the vacuole/lysosome suggested an independent Golgi-based mechanism also at work. Although little is understood of the post-ER pathway, the growing number of variants using it suggests a major role in quality control. Why seemingly redundant mechanisms in sequential compartments are needed is unclear. To understand their physiological relationship, the identification of endogenous pathway-specific substrates is a prerequisite. With ERAD substrates already well characterized, the discovery of Wsc1p as an obligate substrate of Golgi quality control enabled detailed cross-pathway analyses for the first time. By analyzing a panel of engineered substrates, the data show that the surveillance mode is determined by each polypeptide''s intrinsic design. Although most secretory pathway proteins can display ERAD determinants when misfolded, the lack thereof shields Wsc1p from inspection by ER surveillance. Additionally, a powerful ER export signal mediates transport whether the luminal domain is folded or not. By evading ERAD through these passive and active mechanisms, Wsc1p is fully dependent on the post-ER system for its quality control.     

Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways

About 40% of the eukaryotic cell’s proteins are inserted co- or post-translationally in the endoplasmic reticulum (ER), where they attain the native structure under the assistance of resident molecular chaperones and folding enzymes. Subsequently, these proteins are secreted from cells or are transported to their sites of function at the plasma membrane or in organelles of the secretory and endocytic compartments. Polypeptides that are not delivered within the ER (mis-localized proteins, MLPs) are rapidly destroyed by cytosolic proteasomes, with intervention of the membrane protease ZMPSTE24 if they remained trapped in the SEC61 translocation machinery. Proteins that enter the ER, but fail to attain the native structure are rapidly degraded to prevent toxic accumulation of aberrant gene products. The ER does not contain degradative devices and the majority of misfolded proteins generated in this biosynthetic compartment are dislocated across the membrane for degradation by cytosolic 26S proteasomes by mechanisms and pathways collectively defined as ER-associated degradation (ERAD). Proteins that do not engage ERAD factors, that enter aggregates or polymers, are too large, display chimico/physical features that prevent dislocation across the ER membrane (ERAD-resistant misfolded proteins) are delivered to endo-lysosome for clearance, by mechanisms and pathways collectively defined as ER-to-lysosomes-associated degradation (ERLAD). Emerging evidences lead us to propose ERLAD as an umbrella term that includes the autophagic and non-autophagic pathways activated and engaged by ERAD-resistant misfolded proteins generated in the ER for delivery to degradative endo-lysosomes.     

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.     

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

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.