Turnover of EDEM1, an ERAD-enhancing factor, is mediated by multiple degradation routes.

Quality-based protein production and degradation in the endoplasmic reticulum (ER) are essential for eukaryotic cell survival. During protein maturation in the ER, misfolded or unassembled proteins are destined for disposal through a process known as ER-associated degradation (ERAD). EDEM1 is an ERAD-accelerating factor whose gene expression is upregulated by the accumulation of aberrant proteins in the ER, known as ER stress. Although the role of EDEM1 in ERAD has been studied in detail, the turnover of EDEM1 by intracellular degradation machinery, including the proteasome and autophagy, is not well understood. To clarify EDEM1 regulation in the protein level, degradation mechanism of EDEM1 was examined. Our results indicate that both ERAD and autophagy degrade EDEM1 alike misfolded degradation substrates, although each degradation machinery targets EDEM1 in different folded states of proteins. We also found that ERAD factors, including the SEL1L/Hrd1 complex, YOD1, XTP3B, ERdj3, VIMP, BAG6, and JB12, but not OS9, are involved in EDEM1 degradation in a mannose-trimming-dependent and -independent manner. Our results suggest that the ERAD accelerating factor, EDEM1, is turned over by the ERAD itself, similar to ERAD clients.

Lipid biosynthesis perturbation impairs endoplasmic reticulum-associated degradation.

The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein.

Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one-third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determine that TTC17 is a large, soluble, ER-localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and cochaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of insulin-like growth factor type 1 receptor, glycoprotein nonmetastatic melanoma protein B, clusterin, and UDP-glucose:glycoprotein glucosyltransferase 1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins.

The ability of mammalian cells to correctly identify and degrade misfolded secretory proteins, most of them bearing N-glycans, is crucial for their correct function and survival. An inefficient disposal mechanism results in the accumulation of misfolded proteins and consequent endoplasmic reticulum (ER) stress. N-glycan processing creates a code that reveals the folding status of each molecule, enabling continued folding attempts or targeting of the doomed glycoprotein for disposal. We review here the main steps involved in the accurate processing of unfolded glycoproteins. We highlight recent data suggesting that the processing is not stochastic, but that there is selective accelerated glycan trimming on misfolded glycoprotein molecules.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation.

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.

Glycosylation limits forward trafficking of the tetraspan membrane protein PMP22.

Peripheral myelin protein 22 (PMP22) folds and trafficks inefficiently, with only 20% of newly expressed protein trafficking to the cell surface. This behavior is exacerbated in many of the mutants associated with Charcot-Marie-Tooth disease, motivating further study. Here we characterized the role of N-glycosylation in limiting PMP22 trafficking. We first eliminated N-glycosylation using an N41Q mutation, which resulted in an almost 3-fold increase in trafficking efficiency of wildtype (WT) PMP22 and a 10-fold increase for the severely unstable L16P disease mutant in HEK293 cells, with similar results in Schwann cells. Total cellular levels were also much higher for the WT/N41Q mutant, although not for the L16P/N41Q form. Depletion of oligosaccharyltransferase OST-A and OST-B subunits revealed that WT PMP22 is N-glycosylated posttranslationally by OST-B, whereas L16P is cotranslationally glycosylated by OST-A. Quantitative proteomic screens revealed similarities and differences in the interactome for WT, glycosylation-deficient, and unstable mutant forms of PMP22 and also suggested that L16P is sequestered at earlier stages of endoplasmic reticulum quality control. CRISPR knockout studies revealed a role for retention in endoplasmic reticulum sorting receptor 1 (RER1) in limiting the trafficking of all three forms, for UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1) in limiting the trafficking of WT and L16P but not N41Q, and calnexin (CNX) in limiting the trafficking of WT and N41Q but not L16P. This work shows that N-glycosylation is a limiting factor to forward trafficking PMP22 and sheds light on the proteins involved in its quality control.

An autonomous, but INSIG-modulated, role for the sterol sensing domain in mallostery-regulated ERAD of yeast HMG-CoA reductase.

HMG-CoA reductase (HMGR) undergoes feedback-regulated degradation as part of sterol pathway control. Degradation of the yeast HMGR isozyme Hmg2 is controlled by the sterol pathway intermediate GGPP, which causes misfolding of Hmg2, leading to degradation by the HRD pathway; we call this process mallostery. We evaluated the role of the Hmg2 sterol sensing domain (SSD) in mallostery, as well as the involvement of the highly conserved INSIG proteins. We show that the Hmg2 SSD is critical for regulated degradation of Hmg2 and required for mallosteric misfolding of GGPP as studied by in vitro limited proteolysis. The Hmg2 SSD functions independently of conserved yeast INSIG proteins, but its function was modulated by INSIG, thus imposing a second layer of control on Hmg2 regulation. Mutant analyses indicated that SSD-mediated mallostery occurred prior to and independent of HRD-dependent ubiquitination. GGPP-dependent misfolding was still extant but occurred at a much slower rate in the absence of a functional SSD, indicating that the SSD facilitates a physiologically useful rate of GGPP response and implying that the SSD is not a binding site for GGPP. Nonfunctional SSD mutants allowed us to test the importance of Hmg2 quaternary structure in mallostery: a nonresponsive Hmg2 SSD mutant strongly suppressed regulation of a coexpressed, normal Hmg2. Finally, we have found that GGPP-regulated misfolding occurred in detergent-solubilized Hmg2, a feature that will allow next-level analysis of the mechanism of this novel tactic of ligand-regulated misfolding.

Hepatic Sel1L-Hrd1 ER-associated degradation (ERAD) manages FGF21 levels and systemic metabolism via CREBH.

Fibroblast growth factor 21 (Fgf21) is a liver-derived, fasting-induced hormone with broad effects on growth, nutrient metabolism, and insulin sensitivity. Here, we report the discovery of a novel mechanism regulating Fgf21 expression under growth and fasting-feeding. The Sel1L-Hrd1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)-associated degradation (ERAD) machinery. Mice with liver-specific deletion of Sel1L exhibit growth retardation with markedly elevated circulating Fgf21, reaching levels close to those in Fgf21 transgenic mice or pharmacological models. Mechanistically, we show that the Sel1L-Hrd1 ERAD complex controls Fgf21 transcription by regulating the ubiquitination and turnover (and thus nuclear abundance) of ER-resident transcription factor Crebh, while having no effect on the other well-known Fgf21 transcription factor Pparα. Our data reveal a physiologically regulated, inverse correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 levels under fasting-feeding and growth. This study not only establishes the importance of Sel1L-Hrd1 ERAD in the liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic "ERAD-Crebh-Fgf21" axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.

A positive genetic selection for transmembrane domain mutations in HRD1 underscores the importance of Hrd1 complex integrity during ERAD.

Misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for ubiquitination and degradation by the proteasome. During this process, known as ER-associated degradation (ERAD), the ER-embedded Hrd1 ubiquitin ligase plays a central role in recognizing, ubiquitinating, and retrotranslocating scores of lumenal and integral membrane proteins. To better define the mechanisms underlying Hrd1 function in Saccharomyces cerevisiae, several model substrates have been developed. One substrate is Sec61-2, a temperature sensitive allele of the Sec61 translocation channel. Cells expressing Sec61-2 grow at 25 °C because the protein is stable, but sec61-2 yeast are inviable at 38 °C because the mutated protein is degraded in a Hrd1-dependent manner. Therefore, deleting HRD1 stabilizes Sec61-2 and hence sec61-2hrd1∆ double mutants are viable at 38 °C. This unique phenotype allowed us to perform a non-biased screen for loss-of-function alleles in HRD1. Based on its importance in mediating substrate retrotranslocation, the screen was also developed to focus on mutations in sequences encoding Hrd1's transmembrane-rich domain. Ultimately, a group of recessive mutations was identified in HRD1, including an ensemble of destabilizing mutations that resulted in the delivery of Hrd1 to the ERAD pathway. A more stable mutant resided in a buried transmembrane domain, yet the Hrd1 complex was disrupted in yeast expressing this mutant. Together, these data confirm the importance of Hrd1 complex integrity during ERAD, suggest that allosteric interactions between transmembrane domains regulate Hrd1 complex formation, and provide the field with new tools to define the dynamic interactions between ERAD components during substrate retrotranslocation.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 µg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

The Rice Malectin Regulates Plant Cell Death and Disease Resistance by Participating in Glycoprotein Quality Control.

In animals, malectin is well known to play an essential role in endoplasmic reticulum quality control (ERQC) by interacting with ribophorin I, one unit of the oligosaccharyltransferase (OST) complex. However, the functions of malectin in plants remain largely unknown. Here, we demonstrate the rice OsMLD1 is an ER- and Golgi-associated malectin protein and physically interacts with rice homolog of ribophorin I (OsRpn1), and its disruption leads to spontaneous lesion mimic lesions, enhanced disease resistance, and prolonged ER stress. In addition, there are many more N-glycosites and N-glycoproteins identified from the mld1 mutant than wildtype. Furthermore, OsSERK1 and OsSERK2, which have more N-glycosites in mld1, were demonstrated to interact with OsMLD1. OsMLD1 can suppress OsSERK1- or OsSERK2-induced cell death. Thus, OsMLD1 may play a similar role to its mammalian homologs in glycoprotein quality control, thereby regulating cell death and immunity of rice, which uncovers the function of malectin in plants.

Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide.

One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.

Calnexin mediates the maturation of GPI-anchors through ER retention.

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin.

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain.

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum-associated degradation.

The endoplasmic reticulum-associated degradation (ERAD) pathway mediates the endoplasmic reticulum-to-cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K-resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.

SDF2-like protein 1 (SDF2L1) regulates the endoplasmic reticulum localization and chaperone activity of ERdj3 protein.

Molecular chaperones facilitate protein folding by associating with nascent polypeptides, thereby preventing protein misfolding and aggregation. Endoplasmic reticulum (ER) chaperone BiP, the sole HSP70 chaperone in the ER, is regulated by HSP40 chaperones, including ER-resident protein ERdj3 (DNAJB11). ERdj3 lacks an ER retrieval signal, is secreted under ER stress conditions, and functions as a chaperone in the extracellular space, but how its secretion is regulated remains unclear. We recently showed that ERdj3 forms a complex with ER-resident stromal cell-derived factor 2 (SDF2) and SDF2L1 (SDF2-like protein 1) and thereby prevents protein aggregation during the BiP chaperone cycle. However, the contribution of the ERdj3-SDF2L1 complex to protein quality control is poorly understood. Here, we analyzed the intracellular localization and chaperone activity of ERdj3 in complex with SDF2L1. We found that ERdj3 was retained in the ER by associating with SDF2/SDF2L1. In vitro analyses revealed that the ERdj3 dimer incorporated two SDF2L1 molecules; otherwise, ERdj3 alone formed a homotetramer. The ERdj3-SDF2L1 complex suppressed ER protein aggregation, and this suppression did not require substrate transfer to BiP. The ERdj3-SDF2L1 complex inhibited aggregation of denatured GSH S-transferase (GST) in vitro and maintained GST in a soluble oligomeric state. Both in cellulo and in vitro, the chaperone activities of the ERdj3-SDF2L1 complex were higher than those of ERdj3 alone. These findings suggest that, under normal conditions, ERdj3 functions as an ER chaperone in complex with SDF2/SDF2L1 but is secreted into the extracellular space when it cannot form this complex.