Autophagy eliminates a specific species of misfolded procollagen and plays a protective role in cell survival against ER stress

Type I collagen is one of the most abundant proteins in the human body and is essential for tissue formation. Mutations in collagen cause severe abnormalities in bone formation, including osteogenesis imperfecta. Although the mutant collagens are retained in the endoplasmic reticulum (ER) and are toxic to the cell, little is known about how they are removed from the ER. Using two independent cell lines that produce misfolded collagens, we recently demonstrated that procollagen, which is misfolded and accumulated as trimers, is eliminated through the autophagy-lysosomal pathway, not through the ER-associated degradation (ERAD) pathway. In contrast, misfolded procollagen monomer is degraded via ERAD. Moreover, autophagic elimination and ERAD occur independently and exert protective roles and promote cell survival. Thus, autophagy and ERAD, in concert, contribute to eliminating toxic species of misfolded and accumulated proteins from the ER.     

Autophagy: an ER protein quality control process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome-dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both alpha1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.     

Autophagy: an ER Protein Quality Control Process

Protein quality control processes active in the endoplasmic reticulum (ER), including ER-associated protein degradation (ERAD) and the unfolded protein response (UPR), prevent the cytotoxic effects that can result from the accumulation of misfolded proteins. Characterization of a yeast mutant deficient in ERAD, a proteasome–dependent degradation pathway, revealed the employment of two overflow pathways from the ER to the vacuole when ERAD was compromised. One removes the soluble misfolded protein via the biosynthetic pathway and the second clears aggregated proteins via autophagy. Previously, autophagy had been implicated in the clearance of cytoplasmic aggresomes, but was not known to play a direct role in ER protein quality control. These findings provide insight into the molecular mechanisms that result in the gain-of-function liver disease associated with both a1-deficiency and hypofibrinogenemia (abnormally low levels of plasma fibrinogen, which is required for blood clotting), and emphasize the need for a more complete understanding of the molecular mechanisms of autophagy and its relationship to protein quality control.

Addendum to:

Characterization of an ERAD Gene as VPS30/ATG6 Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble A1PiZ and Another for Aggregates of A1PiZ

K.B. Kruse, J.L. Brodsky and A.A. McCracken

Mol Biol Cell 2005; In press.     

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum-associated degradation requires Atg19 and the Cvt/autophagy pathway

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H(+)-ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild-type allele, both proteins are retained in the ER. We have used a pma1-D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1-pma1-D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1-D378T. ATG19 accelerates the degradation of pma1-D378T thus allowing the co-retained wild-type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1.     

A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.     

Glucose-regulated Protein 94 Triage of Mutant Myocilin through Endoplasmic Reticulum-associated Degradation Subverts a More Efficient Autophagic Clearance Mechanism

Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that β-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.     

Multiple endoplasmic reticulum-associated pathways degrade mutant yeast carboxypeptidase Y in mammalian cells

The degradation of misfolded and unassembled proteins by the endoplasmic reticulum (ER)-associated degradation (ERAD) has been shown to occur mainly through the ubiquitin-proteasome pathway after transport of the protein to the cytosol. Recent work has revealed a role for N-linked glycans in targeting aberrant glycoproteins to ERAD. To further characterize the molecular basis of substrate recognition and sorting during ERAD in mammalian cells, we expressed a mutant yeast carboxypeptidase Y (CPY*) in CHO cells. CPY* was retained in the ER in un-aggregated form, and degraded after a 45-min lag period. Degradation was predominantly by a proteasome-independent, non-lysosomal pathway. The inhibitor of ER mannosidase I, kifunensine, blocked the degradation by the alternate pathway but did not affect the proteasomal fraction of degradation. Upon inhibition of glucose trimming, the initial lag period was eliminated and degradation thus accelerated. Our results indicated that, although the proteasome is a major player in ERAD, alternative routes are present in mammalian cells and can play an important role in the disposal of both glycoproteins and non-glycoproteins.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins

Many misfolded endoplasmic reticulum (ER) proteins are eliminated by ERAD, a process in which substrates are polyubiquitylated and moved into the cytosol for proteasomal degradation. We have identified in S. cerevisiae distinct ubiquitin-ligase complexes that define different ERAD pathways. Proteins with misfolded ER-luminal domains use the ERAD-L pathway, in which the Hrd1p/Hrd3p ligase forms a near stoichiometric membrane core complex by binding to Der1p via the linker protein Usa1p. This core complex associates through Hrd3p with Yos9p, a substrate recognition protein in the ER lumen. Substrates with misfolded intramembrane domains define a pathway (ERAD-M) that differs from ERAD-L by being independent of Usa1p and Der1p. Membrane proteins with misfolded cytosolic domains use the ERAD-C pathway and are directly targeted to the Doa10p ubiquitin ligase. All three pathways converge at the Cdc48p ATPase complex. These results lead to a unifying concept for ERAD that may also apply to mammalian cells.     

PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the ER membrane kinases PERK and IRE1 leading to the unfolded protein response (UPR). We show here that UPR activation triggers PERK and IRE1 segregation from BiP and their sorting with misfolded proteins to the ER-derived quality control compartment (ERQC), a pericentriolar compartment that we had identified previously. PERK phosphorylates translation factor eIF2alpha, which then accumulates on the cytosolic side of the ERQC. Dominant negative PERK or eIF2alpha(S51A) mutants prevent the compartmentalization, whereas eIF2alpha(S51D) mutant, which mimics constitutive phosphorylation, promotes it. This suggests a feedback loop where eIF2alpha phosphorylation causes pericentriolar concentration at the ERQC, which in turn amplifies the UPR. ER-associated degradation (ERAD) is an UPR-dependent process; we also find that ERAD components (Sec61beta, HRD1, p97/VCP, ubiquitin) are recruited to the ERQC, making it a likely site for retrotranslocation. In addition, we show that autophagy, suggested to play a role in elimination of aggregated proteins, is unrelated to protein accumulation in the ERQC.     

Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human alpha-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ

The Z variant of human alpha-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins.     

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.     

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.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

The Arabidopsis homolog of the mammalian OS-9 protein plays a key role in the endoplasmic reticulum-associated degradation of misfolded receptor-like kinases

The endoplasmic reticulum-associated degradation (ERAD) is a highly conserved mechanism to remove misfolded membrane/secretory proteins from the endoplasmic reticulum (ER). While many of the individual components of the ERAD machinery are well characterized in yeast and mammals, our knowledge of a plant ERAD process is rather limited. Here, we report a functional study of an Arabidopsis homolog (AtOS9) of an ER luminal lectin Yos9 (OS-9 in mammals) that recognizes a unique asparagine-linked glycan on misfolded proteins. We discovered that AtOS9 is an ER-localized glycoprotein that is co-expressed with many known/predicted ER chaperones. A T-DNA insertional atos9-t mutation blocks the degradation of a structurally imperfect yet biochemically competent brassinosteroid (BR) receptor bri1-9, causing its increased accumulation in the ER and its consequent leakage to the cell surface responsible for restoring the BR sensitivity and suppressing the dwarfism of the bri1-9 mutant. In addition, we identified a missense mutation in AtOS9 in a recently discovered ERAD mutant ems-mutagenized bri1 suppressor 6 (ebs6-1). Moreover, we showed that atos9-t also inhibits the ERAD of bri1-5, another ER-retained BR receptor, and a misfolded EFR, a BRI1-like receptor for the bacterial translation elongation factor EF-Tu. Furthermore, we found that AtOS9 interacted biochemically and genetically with EBS5, an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L known to collaborate with Yos9/OS-9 to select ERAD clients. Taken together, our results demonstrated a functional role of AtOS9 in a plant ERAD process that degrades misfolded receptor-like kinases.     

Activation of the Ras-cAMP signal transduction pathway inhibits the proteasome-independent degradation of misfolded protein aggregates in the endoplasmic reticulum lumen

Many kinds of misfolded secretory proteins are known to be degraded in the endoplasmic reticulum (ER). Dislocation of misfolded proteins from the ER to the cytosol and subsequent degradation by the proteasome have been demonstrated. Using the yeast Saccharomyces cerevisiae, we have been studying the secretion of a heterologous protein, Rhizopus niveus aspartic proteinase-I (RNAP-I). Previously, we found that the pro sequence of RNAP-I is important for the folding and secretion, and that Deltapro, a mutated derivative of RNAP-I in which the entire region of the pro sequence is deleted, forms gross aggregates in the yeast ER. In this study, we show that the degradation of Deltapro occurs independently of the proteasome. Its degradation was not inhibited either by a potent proteasome inhibitor or in a proteasome mutant. We also show that neither the export from the ER nor the vacuolar proteinase is required for the degradation of Deltapro. These results raise the possibility that the Deltapro aggregates are degraded in the ER lumen. We have isolated a yeast mutant in which the degradation of Deltapro is delayed. We show that the mutated gene is IRA2, which encodes a GTPase-activating protein for Ras. Because Ira2 protein is a negative regulator of the Ras-cAMP pathway, this result suggests that hyperactivation of the Ras-cAMP pathway inhibits the degradation of Deltapro. Consistently, down-regulation of the Ras-cAMP pathway in the ira2 mutant suppressed the defect of the degradation of Deltapro. Thus, the Ras-cAMP signal transduction pathway seems to control the proteasome-independent degradation of the ER misfolded protein aggregates.     

BsdABsd2‐dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy

Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non‐endocytic vacuolar turnover of ΔR481 is dependent on BsdA^Bsd2, an ER transmembrane adaptor of HulA^Rsp5 ubiquitin ligase. We obtain in vivo evidence that BsdA^Bsd2 interacts with HulA^Rsp5 and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO^ts) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.     

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.     

Using a ubiquitin ligase as an unfolded protein sensor

A significant fraction of all proteins are misfolded and must be degraded. The ubiquitin-proteasome pathway provides an essential protein quality control function necessary for normal cellular homeostasis. Substrate specificity is mediated by proteins called ubiquitin ligases. In the endoplasmic reticulum (ER) a specialized pathway, the endoplasmic reticulum associated degradation (ERAD) pathway provides means to eliminate misfolded proteins from the ER. One marker used by the ER to identify misfolded glycoproteins is the presence of a high-mannose (Man5-8GlcNAc2) glycan. Recently, FBXO2 was shown to bind high mannose glycans and participate in ERAD. Using glycan arrays, immobilized glycoprotein pulldowns, and glycan competition assays we demonstrate that FBXO2 preferentially binds unfolded glycoproteins. Using recombinant, bacterially expressed GST-FBXO2 as an unfolded protein sensor we demonstrate it can be used to monitor increases in misfolded glycoproteins after physiological or pharmaceutical stressors.