Endoplasmic Reticulum Quality Control Machinery Research Articles

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

Drug resistance mechanisms create targetable proteostatic vulnerabilities in Her2+ breast cancers.

Oncogenic kinase inhibitors show short-lived responses in the clinic due to high rate of acquired resistance. We previously showed that pharmacologically exploiting oncogene-induced proteotoxic stress can be a viable alternative to oncogene-targeted therapy. Here, we performed extensive analyses of the transcriptomic, metabolomic and proteostatic perturbations during the course of treatment of Her2+ breast cancer cells with a Her2 inhibitor covering the drug response, resistance, relapse and drug withdrawal phases. We found that acute Her2 inhibition, in addition to blocking mitogenic signaling, leads to significant decline in the glucose uptake, and shutdown of glycolysis and of global protein synthesis. During prolonged therapy, compensatory overexpression of Her3 allows for the reactivation of mitogenic signaling pathways, but fails to re-engage the glucose uptake and glycolysis, resulting in proteotoxic ER stress, which maintains the protein synthesis block and growth inhibition. Her3-mediated cell proliferation under ER stress during prolonged Her2 inhibition is enabled due to the overexpression of the eIF2 phosphatase GADD34, which uncouples protein synthesis block from the ER stress response to allow for active cell growth. We show that this imbalance in the mitogenic and proteostatic signaling created during the acquired resistance to anti-Her2 therapy imposes a specific vulnerability to the inhibition of the endoplasmic reticulum quality control machinery. The latter is more pronounced in the drug withdrawal phase, where the de-inhibition of Her2 creates an acute surge in the downstream signaling pathways and exacerbates the proteostatic imbalance. Therefore, the acquired resistance mechanisms to oncogenic kinase inhibitors may create secondary vulnerabilities that could be exploited in the clinic.

Identification of two rate-limiting steps in the degradation of partially folded immunoglobulin light chains

Antibody monomers are produced from two immunoglobulin heavy chains and two light chains that are folded and assembled in the endoplasmic reticulum This process is assisted and monitored by components of the endoplasmic reticulum quality control machinery; an outcome made more fraught by the unusual genetic machinations employed to produce a seemingly unlimited antibody repertoire. Proper functioning of the adaptive immune system is as dependent on the success of this operation, as it is on the ability to identify and degrade those molecules that fail to reach their native state. In this study, two rate-limiting steps were identified in the degradation of a non-secreted κ light chain. Both focus on the constant domain (CL), which has evolved to fold rapidly and very stably to serve as a catalyst for the folding of the heavy chain CH1 domain. The first hurdle is the reduction of the disulfide bond in the CL domain, which is required for retrotranslocation to the cytosol. In spite of being reduced, the CL domain retains structure, giving rise to the second rate-limiting step, the unfolding of this domain at the proteasome, which results in a stalled degradation intermediate.

A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor‐mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR‐Cas9 or knockout‐mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER‐resident lectin chaperone Calnexin (CANX) and the ER‐phagy receptor FAM134B are required for autophagy‐mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co‐receptor that recognizes ER luminal misfolded procollagens and interacts with the ER‐phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane‐associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome‐resistant misfolded clients from the ER.

EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α1-antitrypsin ER-associated protein degradation (ERAD) clients Z and NHK, specifically through the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client-binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycan-trimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C terminus of EDEM1 in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5 as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3.

Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.

Peters Plus Syndrome Mutations Disrupt a Noncanonical ER Quality-Control Mechanism

O-fucose is added to cysteine-rich domains called thrombospondin type 1 repeats (TSRs) by protein O-fucosyltransferase 2 (POFUT2) and is elongated with glucose by β3-glucosyltransferase (B3GLCT). Mutations in B3GLCT result in Peters plus syndrome (PPS), an autosomal recessive disorder characterized by eye and other developmental defects. Although 49 putative targets are known, the function of the disaccharide and its role in PPS remain unexplored. Here we show that while POFUT2 is required for secretion of all targets tested, B3GLCT only affects the secretion of a subset, consistent with the observation that B3GLCT mutant phenotypes in PPS patients are less severe than embryonic lethal phenotypes of Pofut2-null mice. O-glycosylation occurs cotranslationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, whereas TSRs from protein still folding in the ER are partially modified, suggesting that O-glycosylation marks folded TSRs and promotes ER exit. In vitro unfolding assays demonstrate that fucose and glucose stabilize folded TSRs in an additive manner. In vitro refolding assays under redox conditions showed that POFUT2 recognizes, glycosylates, and stabilizes the folded form of TSRs, resulting in a net acceleration of folding. While known ER quality-control machinery rely on identifying and tagging unfolded proteins, we find that POFUT2 and B3GLCT mediate a noncanonical ER quality-control mechanism that recognizes folded TSRs and stabilizes them by glycosylation. Our findings provide a molecular basis for the defects observed in PPS and potential targets that contribute to the pathology.

Chemical corrector treatment ameliorates increased seizure susceptibility in a mouse model of familial epilepsy.

Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.

The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1(-/-) mice have reduced viability and fail to thrive early after birth. Male Ube2j1(-/-) mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1(-/-) elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.

When supply does not meet demand-ER stress and plant programmed cell death.

The endoplasmic reticulum (ER) is the central organelle in the eukaryotic secretory pathway. The ER functions in protein synthesis and maturation and is crucial for proper maintenance of cellular homeostasis and adaptation to adverse environments. Acting as a cellular sentinel, the ER is exquisitely sensitive to changing environments principally via the ER quality control machinery. When perturbed, ER-stress triggers a tightly regulated and highly conserved, signal transduction pathway known as the unfolded protein response (UPR) that prevents the dangerous accumulation of unfolded/misfolded proteins. In situations where excessive UPR activity surpasses threshold levels, cells deteriorate and eventually trigger programmed cell death (PCD) as a way for the organism to cope with dysfunctional or toxic signals. The programmed cell death that results from excessive ER stress in mammalian systems contributes to several important diseases including hypoxia, neurodegeneration, and diabetes. Importantly, hallmark features and markers of cell death that are associated with ER stress in mammals are also found in plants. In particular, there is a common, conserved set of chaperones that modulate ER cell death signaling. Here we review the elements of plant cell death responses to ER stress and note that an increasing number of plant-pathogen interactions are being identified in which the host ER is targeted by plant pathogens to establish compatibility.

Approaches to imaging unfolded secretory protein stress in living cells.

The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

Abstract C17: Revealing targetable cancer cell vulnerabilities by in silico pathway profiling

Abstract Successful targeted therapy of cancers relies on comprehensive understanding of vulnerabilities of the tumorigenic platform. Recent cancer genomics efforts have provided with an unprecedented opportunity to gain an unbiased insight into the functional make-up of human cancers. We have devised a computational strategy to leverage cancer genomic datasets to identify candidate pathway vulnerabilities in cancers. The core of our approach is in the delineation of molecular pathways that are activated in tumor cells as a result of secondary selection events during tumor evolution. These secondary pathways are likely to be required for tumorigenesis in the given tumor context. We applied this strategy on HER2+ breast cancers using breast cancer genomic data in the TCGA, the recently published METABRIC and the Cancer Cell Line Encyclopedia (CCLE) datasets. Although HER2+ breast cancers are treated with HER2-targeting agents in the clinic, the response is usually short-lived due to de novo and acquired resistance. We have found that HER2+ breast cancers are characterized by extensive and robust secondary upregulation of the endoplasmic reticulum (ER) quality control (ERQC) pathways, including the ER-associated degradation (ERAD) and PERK/eIF2 pathways. Our follow-up combined in silico and in vitro analyses on a panel of HER2+ and HER2- breast cancer cell lines revealed a model of HER2-driven tumorigenesis where yin-yang interplay between hyperactive HER2/mTOR signaling and the ERQC machinery regulate the ER protein homeostasis to control survival of HER2+ cells. Furthermore, we report that this delicate regulatory architecture can be exploited for therapeutic purposes, such that, combinatorial targeting of ERQC pathways to disturb the proteostatic balance synergized in selectively inducing proteotoxic cell death in therapy-refractory HER2+ cells. This study presents a computational method for identification of cancer pathway vulnerabilities, and suggests that exploiting proteotoxic ER stress may be an effective therapeutic strategy in therapy-refractory HER2+ breast cancers. Note: This abstract was not presented at the conference. Citation Format: Rashika Joshi, Kakajan Komurov. Revealing targetable cancer cell vulnerabilities by in silico pathway profiling. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C17.

The silent codon change I507-ATC->ATT contributes to the severity of the ΔF508 CFTR channel dysfunction.

The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out-of-frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine-508 (ΔF508) and a silent codon change (SCC) for isoleucine-507 (I507-ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum-associated degradation (ERAD). We have demonstrated that the I507-ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507-ATT and I507-ATC ΔF508 CFTR, we establish that the I507-ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR. We demonstrate that the I507-ATC ΔF508 CFTR is less susceptible to the ER quality-control machinery during translation than the I507-ATT, although 27°C correction is necessary for sufficient cell-surface expression. Whole-cell patch-clamp recordings indicate sustained, thermally stable cAMP-activated Cl(-) transport through I507-ATC and unstable function of the I507-ATT ΔF508 CFTR. Single-channel recordings reveal improved gating properties of the I507-ATC compared to I507-ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507-ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.

Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum

Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca(2+) which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca(2+) in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.

Unfolded Protein Responses With or Without Unfolded Proteins?

The endoplasmic reticulum (ER) is the site of secretory protein biogenesis. The ER quality control (QC) machinery, including chaperones, ensures the correct folding of secretory proteins. Mutant proteins and environmental stresses can overwhelm the available QC machinery. To prevent and resolve accumulation of misfolded secretory proteins in the ER, cells have evolved integral membrane sensors that orchestrate the Unfolded Protein Response (UPR). The sensors, Ire1p in yeast and IRE1, ATF6, and PERK in metazoans, bind the luminal ER chaperone BiP during homeostasis. As unfolded secretory proteins accumulate in the ER lumen, BiP releases, and the sensors activate. The mechanisms of activation and attenuation of the UPR sensors have exhibited unexpected complexity. A growing body of data supports a model in which Ire1p, and potentially IRE1, directly bind unfolded proteins as part of the activation process. However, evidence for an unfolded protein-independent mechanism has recently emerged, suggesting that UPR can be activated by multiple modes. Importantly, dysregulation of the UPR has been linked to human diseases including Type II diabetes, heart disease, and cancer. The existence of alternative regulatory pathways for UPR sensors raises the exciting possibility for the development of new classes of therapeutics for these medically important proteins.

Rhomboid Family Pseudoproteases Use the ER Quality Control Machinery to Regulate Intercellular Signaling

SummaryIntramembrane proteolysis governs many cellular control processes, but little is known about how intramembrane proteases are regulated. iRhoms are a conserved subfamily of proteins related to rhomboid intramembrane serine proteases that lack key catalytic residues. We have used a combination of genetics and cell biology to determine that these “pseudoproteases” inhibit rhomboid-dependent signaling by the epidermal growth factor receptor pathway in Drosophila, thereby regulating sleep. iRhoms prevent the cleavage of potential rhomboid substrates by promoting their destabilization by endoplasmic reticulum (ER)-associated degradation; this mechanism has been conserved in mammalian cells. The exploitation of the intrinsic quality control machinery of the ER represents a new mode of regulation of intercellular signaling. Inactive cognates of enzymes are common, but their functions are mostly unclear; our data indicate that pseudoenzymes can readily evolve into regulatory proteins, suggesting that this may be a significant evolutionary mechanism.

Malectin Participates in a Backup Glycoprotein Quality Control Pathway in the Mammalian ER

Malectin is a conserved, endoplasmic reticulum (ER)-resident lectin that recognizes high mannose oligosaccharides displaying terminal glucose residues. Here we show that Malectin is an ER stress-induced protein that selectively associates with glycopolypeptides without affecting their entry and their retention in the Calnexin chaperone system. Analysis of the obligate Calnexin client influenza virus hemagglutinin (HA) revealed that Calnexin and Malectin associated with different timing to different HA conformers and that Malectin associated with misfolded HA. Analysis of the facultative Calnexin clients NHK and α1-antitrypsin (α1AT) revealed that induction of Malectin expression to simulate conditions of ER stress resulted in persistent association between the ER lectin and the model cargo glycoproteins, interfered with processing of cargo-linked oligosaccharides and reduced cargo secretion. We propose that Malectin intervention is activated upon ER stress to inhibit secretion of defective gene products that might be generated under conditions of aberrant functioning of the ER quality control machinery.

Anomalous Diffusion Reports on the Interaction of Misfolded Proteins with the Quality Control Machinery in the Endoplasmic Reticulum

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER.

Role of EBAG9 protein in coat protein complex I‐dependent glycoprotein maturation and secretion processes in tumor cells

Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.

Anomalous Diffusion as a Readout for the Folding Status of Transmembrane Proteins

A multitude of transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded polypeptide chains. In the ER, chaperones supervise their folding process and prevent, by still poorly understood mechanisms, a premature export from the ER.Here, we used Fluorescence Correlation Spectroscopy (FCS) to investigate the interaction of a prototypical transmembrane cargo protein, tsO-45-G, a temperature-sensitive mutant of VSV-G, with the ER quality control machinery in vivo by quantifying the proteins diffusion properties in the ER under various conditions. Our experimental data and accompanying simulations show that the diffusion of unfolded tsO-45-GFP in the ER is strongly anomalous, most likely due to a transient oligomerization with UDP-glucose:glycoprotein glucosyltransferase (UGT1). In contrast, folded tsO-45-G, calnexin-associated unfolded tsO-45-G, or a mutant tsO-45-G with only one glycan are significantly less obstructed in their diffusion behavior.