Yos9p detects and targets misfolded glycoproteins for ER-associated degradation

Endoplasmic reticulum (ER) quality control mechanisms monitor the folding of nascent secretory and membrane polypeptides. Immature molecules are actively retained in the folding compartment whereas proteins that fail to fold are diverted to proteasome-dependent degradation pathways. We report that a key pathway of ER quality control consists of a two-lectin receptor system consisting of Yos9p and Htm1/Mnl1p that recognizes N-linked glycan signals embedded in substrates. This pathway recognizes lumenally oriented determinants of soluble and membrane proteins. Yos9p binds directly to substrates to discriminate misfolded from folded proteins. Substrates displaying cytosolic determinants can be degraded independently of this system. Our studies show that mechanistically divergent systems collaborate to guard against passage and accumulation of misfolded proteins in the secretory pathway.     

Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen

ER-associated degradation (ERAD) of glycoproteins depends on dual recognition of protein misfolding and remodeling of the substrate's N-linked glycans. After recognition, substrates are retrotranslocated to the cytosol for proteasomal degradation. To explore the directionality of this process, we fused a highly stable protein, DHFR, to the N or C terminus of the soluble ERAD substrate CPY* in yeast. Degradation of the C-terminal CPY*-DHFR fusion is markedly slowed and is accompanied by DHFR release in the ER lumen. Thus, folded lumenal domains can impede protein retrotranslocation. The ER lumenal protein Yos9p is required for both release of DHFR and degradation of multiple ERAD substrates. Yos9p forms a complex with substrates and has a sugar binding pocket that is essential for its ERAD function. Nonetheless, substrate recognition persists even when the sugar binding site is mutated or CPY* is unglycosylated. These and other considerations suggest that Yos9p plays a critical role in the bipartite recognition of terminally misfolded glycoproteins.     

Yos9, a control protein for misfolded glycosylated and non-glycosylated proteins in ERAD

The endoplasmic reticulum (ER) is responsible for folding and delivery of secretory proteins to their site of action. One major modification proteins undergo in this organelle is N-glycosylation. Proteins that cannot fold properly will be directed to a process known as endoplasmic reticulum associated degradation (ERAD). Processing of N-glycans generates a signal for ERAD. The lectin Yos9 recognizes the N-glycan signal of misfolded proteins and acts as a gatekeeper for the delivery of these substrates to the cytoplasm for degradation. Presence of Yos9 accelerates degradation of the glycosylated model ERAD substrate CPY?. Here we show that Yos9 has also a control function in degradation of the unglycosylated ERAD substrate CPY?0000. It decelerates its degradation rate.     

A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins

We undertook a growth-based screen exploiting the degradation of CTL*, a chimeric membrane-bound ERAD substrate derived from soluble lumenal CPY*. We screened the Saccharomyces cerevisiae genomic deletion library containing approximately 5000 viable strains for mutants defective in endoplasmic reticulum (ER) protein quality control and degradation (ERAD). Among the new gene products we identified Yos9p, an ER-localized protein previously involved in the processing of GPI anchored proteins. We show that deficiency in Yos9p affects the degradation only of glycosylated ERAD substrates. Degradation of non-glycosylated substrates is not affected in cells lacking Yos9p. We propose that Yos9p is a lectin or lectin-like protein involved in the quality control of N-glycosylated proteins. It may act sequentially or in concert with the ERAD lectin Htm1p/Mnl1p (EDEM) to prevent secretion of malfolded glycosylated proteins and deliver them to the cytosolic ubiquitin-proteasome machinery for elimination.     

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.     

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.     

The EDEM and Yos9p families of lectin-like ERAD factors

Protein quality control pathways monitor the folding of newly synthesized proteins throughout the cell. Irreversibly misfolded proteins are sorted and degraded to neutralize their potential toxicity. In the secretory pathway, multiple strategies have evolved to test the wide diversity of molecules that traffic through the endoplasmic reticulum. The organelle has adapted the use of N-linked glycans to signal protein folding states. The signals are read by the EDEM and Yos9 protein families that take substrates out of folding cycles for degradation.     

Structural basis for oligosaccharide recognition of misfolded glycoproteins by OS-9 in ER-associated degradation

Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9(MRH)) complexed with α3,α6-mannopentaose. The OS-9(MRH) has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.     

Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein

The conversion of cellular prion protein (PrP(C)) to the disease-associated misfolded isoform (PrP(Sc)) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrP(Sc) is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism. Here, serial transmission PMCA experiments were compared with serial transmission reactions lacking the sonication steps. We achieved approximately 200,000-fold PrPres amplification by PMCA. In contrast, although initial amplification was comparable to PMCA reactions, PrPres levels quickly dropped below detection limit when samples were not subjected to ultrasound. These results indicate that aggregate breakage is essential for efficient autocatalytic amplification of misfolded prion protein and suggest an important role of aggregate breakage in prion propagation.     

Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD

A functional unfolded protein response (UPR) is essential for endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded secretory proteins, reflecting the fact that some level of UPR activation must exist under normal physiological conditions. A coordinator of the UPR and ERAD processes has long been sought. We previously showed that the PKR-like, ER-localized eukaryotic translation initiation factor 2α kinase branch of the UPR is required for the recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ER-derived quality control compartment (ERQC), a staging ground for ERAD. Here we show that homocysteine-induced ER protein (Herp), a protein highly upregulated by this UPR branch, is responsible for this compartmentalization. Herp localizes to the ERQC, and our results suggest that it recruits HRD1, which targets to ERAD the substrate presented by the OS-9 lectin at the ERQC. Predicted overall structural similarity of Herp to the ubiquitin-proteasome shuttle hHR23, but including a transmembrane hairpin, suggests that Herp may function as a hub for membrane association of ERAD machinery components, a key organizer of the ERAD complex.     

Endoplasmic reticulum (ER)-associated degradation of misfolded N-linked glycoproteins is suppressed upon inhibition of ER mannosidase I

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma-carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.     

Intracellular pathways of folded and misfolded amyloid precursor protein degradation

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein beta-amyloid (Abeta). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when alpha-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.     

Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function

The regulation and homeostasis of autophagy are essential for maintaining organ morphology and function. As a lysosomal membrane protein, the effect of Sidt2 on kidney structure and renal autophagy is still unknown. In this study, we found that the kidneys of Sidt2^−/− mice showed changes in basement membrane thickening, foot process fusion, and mitochondrial swelling, suggesting that the structure of the kidney was damaged. Increased urine protein at 24 h indicated that the kidney function was also damaged. At the same time, the absence of Sidt2 caused a decrease in the number of acidic lysosomes, a decrease in acid hydrolase activity and expression in the lysosome, and an increase of pH in the lysosome, suggesting that lysosomal function was impaired after Sidt2 deletion. The accumulation of autophagolysosomes, increased LC3-II and P62 protein levels, and decreased P62 mRNA levels indicated that the absence of the Sidt2 gene caused abnormal autophagy pathway flow. Chloroquine experiment, immunofluorescence autophagosome, and lysosome fusion assay, and Ad-mcherry-GFP-LC3B further indicated that, after Sidt2 deletion, the production of autophagosomes did not increase, but the fusion of autophagosomes and lysosomes and the degradation of autophagolysosomes were impaired. When incubating Sidt2^−/− cells with the autophagy activator rapamycin, we found that it could activate autophagy, which manifested as an increase in autophagosomes, but it could not improve autophagolysosome degradation. Meanwhile, it further illustrated that the Sidt2 gene plays an important role in the smooth progress of autophagolysosome processes. In summary, the absence of the Sidt2 gene caused impaired lysosome function and a decreased number of acidic lysosomes, leading to formation and degradation disorders of the autophagolysosomes, which eventually manifested as abnormal kidney structure and function. Sidt2 is essential in maintaining the normal function of the lysosomes and the physiological stability of the kidneys.Subject terms: Macroautophagy, Kidney diseases     

Yos9p assists in the degradation of certain nonglycosylated proteins from the endoplasmic reticulum

The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.     

DER7, encoding alpha-glucosidase I is essential for degradation of malfolded glycoproteins of the endoplasmic reticulum

Proteins entering the endoplasmic reticulum (ER) have to acquire an export-competent structure before they are delivered to their final destination. This folding process is monitored by an ER protein quality control system. Folding-incompetent conformers are eliminated via a mechanism called ER-associated protein degradation (ERAD). Genetic studies in the yeast Saccharomyces cerevisiae have revealed that carbohydrate modification plays a crucial role in these processes. Here we show that a previously isolated der mutant (der7-1) is defective in ERAD. We identify DER7 as the gene encoding N-glycan-processing alpha-glucosidase I (EC 3.2.1.106) of the ER and demonstrate that its inactivity, due to a substitution of the conserved glycine residue at position 725 by arginine (G725R) in the der7-1 mutant, leads to ER-stress.     

Unraveling the function of Arabidopsis thaliana OS9 in the endoplasmic reticulum-associated degradation of glycoproteins

In the endoplasmic reticulum, immature polypeptides coincide with terminally misfolded proteins. Consequently, cells need a well-balanced quality control system, which decides about the fate of individual proteins and maintains protein homeostasis. Misfolded and unassembled proteins are sent for destruction via the endoplasmic reticulum-associated degradation (ERAD) machinery to prevent the accumulation of potentially toxic protein aggregates. Here, we report the identification of Arabidopsis thaliana OS9 as a component of the plant ERAD pathway. OS9 is an ER-resident glycoprotein containing a mannose-6-phosphate receptor homology domain, which is also found in yeast and mammalian lectins involved in ERAD. OS9 fused to the C-terminal domain of YOS9 can complement the ERAD defect of the corresponding yeast Δyos9 mutant. An A. thaliana OS9 loss-of-function line suppresses the severe growth phenotype of the bri1-5 and bri1-9 mutant plants, which harbour mutated forms of the brassinosteroid receptor BRI1. Co-immunoprecipitation studies demonstrated that OS9 associates with Arabidopsis SEL1L/HRD3, which is part of the plant ERAD complex and with the ERAD substrates BRI1-5 and BRI1-9, but only the binding to BRI1-5 occurs in a glycan-dependent way. OS9-deficiency results in activation of the unfolded protein response and reduces salt tolerance, highlighting the role of OS9 during ER stress. We propose that OS9 is a component of the plant ERAD machinery and may act specifically in the glycoprotein degradation pathway.     

Structural and biochemical basis of Yos9 protein dimerization and possible contribution to self-association of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation ubiquitin-ligase complex

In yeast, the membrane-bound HMG-CoA reductase degradation (HRD) ubiquitin-ligase complex is a key player of the ER-associated protein degradation pathway that targets misfolded proteins for proteolysis. Yos9, a component of the luminal submodule of the ligase, scans proteins for specific oligosaccharide modifications, which constitute a critical determinant of the degradation signal. Here, we report the crystal structure of the Yos9 domain that was previously suggested to confer binding to Hrd3, another component of the HRD complex. We observe an αβ-roll domain architecture and a dimeric assembly which are confirmed by analytical ultracentrifugation of both the crystallized domain and full-length Yos9. Our binding studies indicate that, instead of this domain, the N-terminal part of Yos9 including the mannose 6-phosphate receptor homology domain mediates the association with Hrd3 in vitro. Our results support the model of a dimeric state of the HRD complex and provide first-time evidence of self-association on its luminal side.     

ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C

Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. In this study, genes specifically induced in response to transient expression of two disease-associated mutations were identified by microarray analyses. Immunoglobulin heavy chain binding protein (BiP) and two heat shock protein 40 family members, endoplasmic reticulum-localized DnaJ homologues ERdj4 and ERdj5, were significantly elevated and exhibited prolonged and specific association with the misfolded proprotein; in contrast, ERdj3 interacted with BiP, but it did not associate with either wild-type or mutant SP-C. Misfolded SP-C, ERdj4, and ERdj5 coprecipitated with p97/VCP indicating that the cochaperones remain associated with the misfolded proprotein until it is dislocated to the cytosol. Knockdown of ERdj4 and ERdj5 expression increased ER retention and inhibited degradation of misfolded SP-C, but it had little effect on the wild-type protein. Transient expression of ERdj4 and ERdj5 in X-box binding protein 1(-/-) mouse embryonic fibroblasts substantially restored rapid degradation of mutant SP-C proprotein, whereas transfection of HPD mutants failed to rescue SP-C endoplasmic reticulum-associated protein degradation. ERdj4 and ERdj5 promote turnover of misfolded SP-C and this activity is dependent on their ability to stimulate BiP ATPase activity.     

Monovalent lectin as a novel tool for the resolution of microheterogeneity in glycoproteins

We have devised a method for the determination of the long-term (days) uptake of radiolabeled surfactants by intact mammalian cells in monolayer culture. We have utilized the method to investigate several characteristics of the uptakes of three highly purified synthetic zwitterionic surfactants by confluent hamster embryo fibroblasts in culture medium containing serum. The uptakes reach a steady state after several hours and appear to be nonsaturable at sublytic concentrations. Surfactant structure was found to greatly influence the rate and extent of cellular uptake as well as the extent of the in vitro surfactant biotransformation.