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1.  ERLIN2 promotes breast cancer cell survival by modulating endoplasmic reticulum stress pathways 
BMC Cancer  2012;12:225.
Background
Amplification of the 8p11-12 region has been found in approximately 15% of human breast cancer and is associated with poor prognosis. Previous genomic analysis has led us to identify the endoplasmic reticulum (ER) lipid raft-associated 2 (ERLIN2) gene as one of the candidate oncogenes within the 8p11-12 amplicon in human breast cancer, particularly in the luminal subtype. ERLIN2, an ER membrane protein, has recently been identified as a novel mediator of ER-associated degradation. Yet, the biological roles of ERLIN2 and molecular mechanisms by which ERLIN2 coordinates ER pathways in breast carcinogenesis remain unclear.
Methods
We established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A) using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for in vitro transforming phenotypes. Next, we knocked down the ERLIN2 as well as the ER stress sensor IRE1α activity in the breast cancer cell lines to characterize the biological roles and molecular basis of the ERLIN2 in carcinogenesis. Finally, immunohistochemical staining was performed to detect ERLIN2 expression in normal and cancerous human breast tissues
Results
We found that amplification of the ERLIN2 gene and over expression of the ERLIN2 protein occurs in both luminal and Her2 subtypes of breast cancer. Gain- and loss-of-function approaches demonstrated that ERLIN2 is a novel oncogenic factor associated with the ER stress response pathway. The IRE1α/XBP1 axis in the ER stress pathway modulated expression of ERLIN2 protein levels in breast cancer cells. We also showed that over expression of ERLIN2 facilitated the adaptation of breast epithelial cells to ER stress by supporting cell growth and protecting the cells from ER stress-induced cell death.
Conclusions
ERLIN2 may confer a selective growth advantage for breast cancer cells by facilitating a cytoprotective response to various cellular stresses associated with oncogenesis. The information provided here sheds new light on the mechanism of breast cancer malignancy
doi:10.1186/1471-2407-12-225
PMCID: PMC3732090  PMID: 22681620
Gene amplification; Breast cancer; Endoplasmic reticulum; ERLIN2
2.  Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells 
The Biochemical journal  2012;446(3):415-425.
Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2 in regulating cytosolic lipid content, down-regulation of ERLIN2 in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2 in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer.
doi:10.1042/BJ20112050
PMCID: PMC3806481  PMID: 22690709
cancer; endoplasmic reticulum (ER); ER membrane lipid raft-associated 2 (ERLIN2); lipogenesis; oncogenesis
3.  WHEN WORLDS COLLIDE: IP3 RECEPTORS AND THE ERAD PATHWAY 
Cell calcium  2009;46(3):147-153.
Summary
While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca2+ store, those who study protein synthesis tend see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP3) receptors are activated, since in addition to acting as release channels for stored ER Ca2+, IP3 receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP3 receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP3 receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP3 receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP3 receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca2+ stores to IP3 and may protect cells against deleterious effects of over-activation of Ca2+ signaling pathways.
doi:10.1016/j.ceca.2009.05.002
PMCID: PMC2752845  PMID: 19709743
4.  CLUSTERED HYDROPHOBIC AMINO ACIDS IN AMPHIPATHIC HELICES MEDIATE ERLIN1/2 COMPLEX ASSEMBLY 
Erlin1 and erlin2 are highly homologous, ~ 40kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of ~2MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the “assembly domain”, that mediates higher-order assembly into ~2MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into ~2MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the ~2MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.
doi:10.1016/j.bbrc.2011.10.032
PMCID: PMC3242435  PMID: 22020079
erlin1; erlin2; amphipathic helix; assembly; crosslinking
5.  A nullimorphic ERLIN2 mutation defines a complicated hereditary spastic paraplegia locus (SPG18) 
Neurogenetics  2011;12(4):333-336.
Hereditary Spastic Paraplegia (HSP) is a clinically and genetically heterogeneous group of neurological disorders that are characterized by progressive spasticity of the lower extremities. We describe an extended consanguineous Saudi family in which HSP is linked to SPG18, a previously reported autosomal recessive locus, and show that it is associated with a nullimorphic deletion of ERLIN2, a component of endoplasmic reticulum associated degradation. This finding adds to the growing diversity of cellular functions that are now known to be involved in the maintenance of the corticospinal tract neurons.
doi:10.1007/s10048-011-0291-8
PMCID: PMC3215864  PMID: 21796390
ERAD; Aphasia; Intellectual disability; ERLIN2
6.  SPFH1 and SPFH2 Mediate the Ubiquitination and Degradation of Inositol 1,4,5-Trisphosphate Receptors in Muscarinic Receptor-Expressing Hela Cells 
Biochimica et biophysica acta  2009;1793(11):1710-1718.
Summary
Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP3 receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP3 receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP3 receptor ERAD in this cell type, and show that IP3 receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP3 receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP3 receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP3 receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IκBα processing, indicating that the SPFH1/2 complex is a key player in IP3 receptor ERAD, acting at a step after IP3 receptor activation, but prior to IP3 receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP3 receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP3 receptors.
doi:10.1016/j.bbamcr.2009.09.004
PMCID: PMC2764810  PMID: 19751772
inositol 1,4,5-trisphosphate receptor; SPFH1; SPFH2; endoplasmic reticulum-associated degradation; ubiquitin; proteasome
7.  Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD 
The Journal of Cell Biology  2009;187(2):201-217.
Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms.
Unwanted proteins in the endoplasmic reticulum (ER) are exported into the cytoplasm and degraded by the proteasome through the ER-associated protein degradation pathway (ERAD). Disturbances in ERAD are linked to ER stress, which has been implicated in the pathogenesis of several human diseases. However, the composition and organization of ERAD complexes in human cells is still poorly understood. In this paper, we describe a trimeric complex that we propose functions in ERAD. Knockdown of erasin, a platform for p97/VCP and ubiquilin binding, or knockdown of ubiquilin in human cells slowed degradation of two classical ERAD substrates. In Caenorhabditis elegans, ubiquilin and erasin are ER stress-response genes that are regulated by the ire-1 branch of the unfolded protein response pathway. Loss of ubiquilin or erasin resulted in activation of ER stress, increased accumulation of polyubiquitinated proteins, and shortened lifespan in worms. Our results strongly support a role for this complex in ERAD and in the regulation of ER stress.
doi:10.1083/jcb.200903024
PMCID: PMC2768832  PMID: 19822669
8.  A Pro-Cathepsin L Mutant Is a Luminal Substrate for Endoplasmic-Reticulum-Associated Degradation in C. elegans 
PLoS ONE  2012;7(7):e40145.
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.
doi:10.1371/journal.pone.0040145
PMCID: PMC3388072  PMID: 22768338
9.  Huntingtin Interacts with the Cue Domain of gp78 and Inhibits gp78 Binding to Ubiquitin and p97/VCP 
PLoS ONE  2010;5(1):e8905.
Huntington's disease (HD) is caused by polyglutamine expansion in huntingtin (htt) protein, but the exact mechanism of HD pathogenesis remains uncertain. Recent evidence suggests that htt proteins with expanded polyglutamine tracts induce endoplasmic reticulum (ER) stress, probably by interfering with ER-associated degradation (ERAD). Here we report that mutant htt interacts and interferes with the function of gp78, an ER membrane-anchored ubiquitin ligase (E3) involved in ERAD. Mapping studies showed that the HEAT repeats 2&3 of htt interact with the cue domain of gp78. The interaction competitively reduces polyubiquitinated protein binding to gp78 and also sterically blocks gp78 interaction of p97/VCP, a molecular chaperone that is essential for ERAD. These effects of htt negatively regulate the function of gp78 in ERAD and are aggravated by polyglutamine expansion. Paradoxically, gp78 is still able to ubiquitinate and facilitate degradation of htt proteins with expanded polyglutamine. The impairment of ERAD by mutant htt proteins is associated with induction of ER stress. Our studies provide a novel molecular mechanism that supports the involvement of ER stress in HD pathogenesis.
doi:10.1371/journal.pone.0008905
PMCID: PMC2811200  PMID: 20126661
10.  RNF-121 Is an Endoplasmic Reticulum-Membrane E3 Ubiquitin Ligase Involved in the Regulation of β-Integrin 
Molecular Biology of the Cell  2010;21(11):1788-1798.
RNF-121 is an E3 ligase RING finger protein that is localized to the ER in Caenorhabditis elegans and functions in the UPR and ERAD pathways. The β subunit of the heterodimeric integrin receptor was identified as a substrate for RNF-121, suggesting a link between ERAD and cell adhesion through the regulation of β-integrin.
We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans. Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the β subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.
doi:10.1091/mbc.E09-09-0774
PMCID: PMC2877638  PMID: 20357004
11.  The Arabidopsis Homolog of the Mammalian OS-9 Protein Plays a Key Role in the Endoplasmic Reticulum-Associated Degradation of Misfolded Receptor-Like Kinases 
Molecular Plant  2012;5(4):929-940.
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.
doi:10.1093/mp/sss042
PMCID: PMC3399701  PMID: 22516478
brassinosteroid receptor; ER quality control; EMS-mutagenized bri1-9 suppressor; lectin; N-glycan; MRH domain
12.  Transforming properties of 8p11-12 amplified genes in human breast cancer 
Cancer research  2010;70(21):8487-8497.
Amplification of the 8p11-12 region has been found in about 15% of human breast cancer and is associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon to identify candidate oncogenes in breast cancer. We identified 21 candidate genes and provided evidence that three genes; LSM-1, TC-1 and BAG4 have transforming properties when over expressed. In the present study, we systematically investigated the transforming properties of thirteen newly identified 8p11-12 candidate oncogenes in vitro. We found that WHSC1L1, DDHD2 and ERLIN2 are the most potently transforming oncogenes we tested from the 8p11-12 region based on the number of altered phenotypes expressed by the cells. WHSC1L1 contains a PWWP-domain that is a methyl-lysine recognition motif involved in histone code modification and epigenetic regulation of gene expression. Knock-down of WHSC1L1 in 8p11-12 amplified breast cancer cells resulted in profound loss of growth and survival of these cells. Further, we identified several WHSC1L1 target genes, one of which is iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family.
doi:10.1158/0008-5472.CAN-10-1013
PMCID: PMC3089754  PMID: 20940404
gene amplification; breast cancer; WHSC1L1
13.  AAA-ATPase p97/Cdc48p, a Cytosolic Chaperone Required for Endoplasmic Reticulum-Associated Protein Degradation 
Molecular and Cellular Biology  2002;22(2):626-634.
Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones. Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response. We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.
doi:10.1128/MCB.22.2.626-634.2002
PMCID: PMC139744  PMID: 11756557
14.  Usa1 Protein Facilitates Substrate Ubiquitylation through Two Separate Domains 
PLoS ONE  2009;4(10):e7604.
Background
Defects in protein folding are recognized as the root of many neurodegenerative disorders. In the endoplasmic reticulum (ER), secretory proteins are subjected to a stringent quality control process to eliminate misfolded proteins by the ER-associated degradation (ERAD) pathway. A novel ERAD component Usa1 was recently identified. However, the specific role of Usa1 in ERAD remains obscure.
Methodology/Principal Findings
Here, we demonstrate that Usa1 is important for substrate ubiquitylation. Furthermore, we defined key cis-elements of Usa1 essential for its degradation function. Interestingly, a putative proteasome-binding motif is dispensable for the functioning of Usa1 in ERAD. We identify two separate cytosolic domains critical for Usa1 activity in ERAD, one of which is involved in binding to the Ub-protein ligase Hrd1/Hrd3. Usa1 may have another novel role in substrate ubiquitylation that is separate from the Hrd1 association.
Conclusions/Significance
We conclude that Usa1 has two important roles in ERAD substrate ubiquitylation.
doi:10.1371/journal.pone.0007604
PMCID: PMC2764048  PMID: 19898607
15.  Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production 
PLoS ONE  2012;7(3):e34169.
Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections.
doi:10.1371/journal.pone.0034169
PMCID: PMC3312915  PMID: 22461906
16.  TorsinA participates in endoplasmic reticulum-associated degradation 
Nature communications  2011;2:393.
TorsinA is an AAA+ ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508) was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VIMP, and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing CFTRΔF508, and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state.
doi:10.1038/ncomms1383
PMCID: PMC3529909  PMID: 21750546
dystonia; movement disorder; secretory pathway; retro-translocation; protein degradation; proteosome; cystic fibrosis; cholera toxin
17.  Stress Tolerance of Misfolded Carboxypeptidase Y Requires Maintenance of Protein Trafficking and Degradative Pathways 
Molecular Biology of the Cell  2003;14(7):2756-2767.
The accumulation of aberrantly folded proteins can lead to cell dysfunction and death. Currently, the mechanisms of toxicity and cellular defenses against their effects remain incompletely understood. In the endoplasmic reticulum (ER), stress caused by misfolded proteins activates the unfolded protein response (UPR). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes to maintain cellular homeostasis. We studied the effects of ER stress in budding yeast through expression of the well-characterized misfolded protein, CPY*. By challenging cells within their physiological limits to resist stress, we show that the UPR is required to maintain essential functions including protein translocation, glycosylation, degradation, and transport. Under stress, the ER-associated degradation (ERAD) pathway for misfolded proteins is saturable. To maintain homeostasis, an “overflow” pathway dependent on the UPR transports excess substrate to the vacuole for turnover. The importance of this pathway was revealed through mutant strains compromised in the vesicular trafficking of excess CPY*. Expression of CPY* at levels tolerated by wild-type cells was toxic to these strains despite retaining the ability to activate the UPR.
doi:10.1091/mbc.E02-11-0717
PMCID: PMC165674  PMID: 12857862
18.  Sec61p Is Required for ERAD-L 
The Journal of Biological Chemistry  2008;283(49):33883-33888.
Misfolded proteins in the endoplasmic reticulum (ER) are exported to the cytosol for degradation by the proteasome in a process known as ER-associated degradation (ERAD). CPY* is a well characterized ERAD substrate whose degradation is dependent upon the Hrd1 complex. However, although the functions of some of the components of this complex are known, the nature of the protein dislocation channel remains obscure. Sec61p has been suggested as an obvious candidate because of its role as a protein-conducting channel through which polypeptides are initially translocated into the ER. However, it has not yet been possible to functionally dissect any role for Sec61p in dislocation from its essential function in translocation. By changing the translocation properties of a series of novel ERAD substrates, we are able to separate these two events and find that functional Sec61p is essential for the ERAD-L pathway.
doi:10.1074/jbc.M803054200
PMCID: PMC2590686  PMID: 18819915
19.  COPII machinery cooperates with ER-localized Hsp40 to sequester misfolded membrane proteins into ER-associated compartments 
Molecular Biology of the Cell  2013;24(5):633-642.
Certain transmembrane ERAD substrates are segregated into specialized ER subdomains, termed ER-associated compartments (ERACs), before degradation. COPII components and Hsp40s act in the same pathway to sequester ERAD substrates into ERACs. The findings point to an as-yet-undefined role of COPII proteins in the formation of ERACs.
Proteins that fail to fold in the endoplasmic reticulum (ER) are subjected to ER-associated degradation (ERAD). Certain transmembrane ERAD substrates are segregated into specialized ER subdomains, termed ER-associated compartments (ERACs), before targeting to ubiquitin–proteasome degradation. The traffic-independent function of several proteins involved in COPII-mediated ER-to-Golgi transport have been implicated in the segregation of exogenously expressed human cystic fibrosis transmembrane conductance regulator (CFTR) into ERACs in Saccharomyces cerevisiae. Here we focus on the properties of COPII components in the sequestration of enhanced green fluorescent protein (EGFP)–CFTR into ERACs. It has been demonstrated that the temperature-sensitive growth defects in many COPII mutants can be suppressed by overexpressing other genes involved in COPII vesicle formation. However, we show that these suppression abilities are not always correlated with the ability to rescue the ERAC formation defect, suggesting that COPII-mediated EGFP-CFTR entry into ERACs is independent of its ER-to-Golgi trafficking function. In addition to COPII machinery, we find that ER-associated Hsp40s are also involved in the sequestration process by directly interacting with EGFP-CFTR. COPII components and ER-associated Hsp40, Hlj1p, act in the same pathway to sequester EGFP-CFTR into ERACs. Our findings point to an as-yet-undefined role of COPII proteins in the formation of ERACs.
doi:10.1091/mbc.E12-08-0639
PMCID: PMC3583666  PMID: 23303252
20.  N-terminal acetylation of the yeast Derlin Der1 is essential for Hrd1 ubiquitin-ligase activity toward luminal ER substrates 
Molecular Biology of the Cell  2013;24(7):890-900.
Up to 70% of yeast proteins are N-terminally acetylated, but in few cases is the function known. The NatB Nα-acetyltransferase is essential for ER-associated degradation of luminal proteins (ERAD-L). Der1, an ERAD-L cofactor of the Hrd1 ubiquitin ligase, is acetylated by NatB and is the only N-acetylation substrate crucial to ERAD-L.
Two conserved ubiquitin ligases, Hrd1 and Doa10, mediate most endoplasmic reticulum–associated protein degradation (ERAD) in yeast. Degradation signals (degrons) recognized by these ubiquitin ligases remain poorly characterized. Doa10 recognizes the Deg1 degron from the MATα2 transcription factor. We previously found that deletion of the gene (NAT3) encoding the catalytic subunit of the NatB N-terminal acetyltransferase weakly stabilized a Deg1-fusion protein. By contrast, a recent analysis of several MATα2 derivatives suggested that N-terminal acetylation of these proteins by NatB was crucial for recognition by Doa10. We now analyze endogenous MATα2 degradation in cells lacking NatB and observe minimal perturbation relative to wild-type cells. However, NatB mutation strongly impairs degradation of ER-luminal Hrd1 substrates. This unexpected defect derives from a failure of Der1, a Hrd1 complex subunit, to be N-terminally acetylated in NatB mutant yeast. We retargeted Der1 to another acetyltransferase to show that it is the only ERAD factor requiring N-terminal acetylation. Preventing Der1 acetylation stimulates its proteolysis via the Hrd1 pathway, at least partially accounting for the ERAD defect observed in the absence of NatB. These results reveal an important role for N-terminal acetylation in controlling Hrd1 ligase activity toward a specific class of ERAD substrates.
doi:10.1091/mbc.E12-11-0838
PMCID: PMC3608499  PMID: 23363603
21.  The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD 
Molecular Biology of the Cell  2007;18(5):1670-1682.
RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
doi:10.1091/mbc.E06-03-0248
PMCID: PMC1855009  PMID: 17314412
22.  Endoplasmic Reticulum Degradation Requires Lumen to Cytosol Signaling 
The Journal of Cell Biology  2000;151(1):69-82.
Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. We show that these two proteins directly interact through the Hrd1p transmembrane domain, allowing Hrd1p stability by Hrd3p-dependent control of the Hrd1p RING-H2 domain activity. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. An engineered, completely lumenal, truncated version of Hrd3p functioned normally in both ERAD and Hrd1p stabilization, indicating that the lumenal domain of Hrd3p regulates the cytosolic Hrd1p RING-H2 domain by signaling through the Hrd1p transmembrane domain. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. The HRD complex engages in lumen to cytosol communication required for regulation of Hrd1p stability and the coordination of ERAD events on both sides of the ER membrane.
PMCID: PMC2189800  PMID: 11018054
proteasome; endoplasmic reticulum; ubiquitin ligase; transmembrane signaling; topology
23.  USP13 antagonizes gp78 to maintain functionality of a chaperone in ER-associated degradation 
eLife  2014;3:e01369.
Physiological adaptation to proteotoxic stress in the endoplasmic reticulum (ER) requires retrotranslocation of misfolded proteins into the cytoplasm for ubiquitination and elimination by ER-associated degradation (ERAD). A surprising paradox emerging from recent studies is that ubiquitin ligases (E3s) and deubiquitinases (DUBs), enzymes with opposing activities, can both promote ERAD. Here we demonstrate that the ERAD E3 gp78 can ubiquitinate not only ERAD substrates, but also the machinery protein Ubl4A, a key component of the Bag6 chaperone complex. Remarkably, instead of targeting Ubl4A for degradation, polyubiquitination is associated with irreversible proteolytic processing and inactivation of Bag6. Importantly, we identify USP13 as a gp78-associated DUB that eliminates ubiquitin conjugates from Ubl4A to maintain the functionality of Bag6. Our study reveals an unexpected paradigm in which a DUB prevents undesired ubiquitination to sharpen substrate specificity for an associated ubiquitin ligase partner and to promote ER quality control.
DOI: http://dx.doi.org/10.7554/eLife.01369.001
eLife digest
Cells make proteins inside a structure called the endoplasmic reticulum. However, some of these proteins cannot fold into the correct shape, so cells rely on a process called the ERAD pathway to degrade and eliminate these faulty proteins. First, however, the misfolded proteins must be moved from the endoplasmic reticulum to the main body of the cell (the cytosol).
The process by which the misfolded proteins are moved through the membrane that encloses the endoplasmic reticulum is complex, with ‘ERAD machinery proteins’ playing an important role. Among them, a series of enzymes called E3 ligases ‘tag’ the faulty proteins with a small protein called ubiquitin, and a complex called the proteasome then recognizes and degrades those proteins that have been tagged with ubiquitin. However, it is not clear why the E3 ligases that tag the misfolded proteins with ubiquitin don’t also tag the machinery proteins that from complexes with the faulty proteins.
Now Liu et al. have used a combination of biochemical and genetic tools to shed light on this puzzle by studying the interaction of gp78—which is an E3 ligase—and USP13, an enzyme that opposes the actions of the E3 ligases by removing ubiquitin. Liu et al. showed that gp78 can indeed tag certain machinery proteins with ubiquitin, which would stop the removal of misfolded proteins from the endoplasmic reticulum. However, USP13 opposed the action of gp78, thus allowing the removal to continue.
It has been known for some time that enzymes with opposing roles—the addition and removal of ubiquitin—can work together, but the biological significance of this phenomenon was not fully understood. The work of Liu et al. suggests that USP13 makes the elimination of misfolded proteins more efficient by ensuring that gp78 only tags those proteins that are misfolded: it does this by removing ubiquitin from proteins that should not have been tagged. A similar phenomenon is known to occur in genetics during DNA replication, with the enzyme complex that replicates the DNA including an enzyme that performs a proofreading role.
DOI: http://dx.doi.org/10.7554/eLife.01369.002
doi:10.7554/eLife.01369
PMCID: PMC3889402  PMID: 24424410
ubiquitin; ERAD; deubiquitinase; ubiquitin ligase; Bag6; gp78; Human
24.  The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance 
Cell Research  2010;21(6):957-969.
Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca2+ release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.
doi:10.1038/cr.2010.181
PMCID: PMC3203697  PMID: 21187857
salt stress; ERAD; UPR; Ca2+; ROS
25.  Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM) 
Cell Stress & Chaperones  2010;15(5):619-629.
Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.
doi:10.1007/s12192-010-0174-1
PMCID: PMC3006622  PMID: 20186508
Endoplasmic reticulum (ER); Mitochondria; Mitochondria-associated membrane (MAM); Oxidative protein folding; Ero1α

Results 1-25 (924972)