GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径

目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein, GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation, ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS-HRD1-KO中的蛋白质积累量(I_PHK)和PIGS-GP78-KO中的蛋白质积累量(I_PG...     

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.     

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis

In mutant INS gene–induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.     

Substrate recognition in ER-associated degradation mediated by Eps1, a member of the protein disulfide isomerase family

Pma1-D378N is a misfolded plasma membrane protein in yeast that is prevented from delivery to the cell surface and targeted instead for ER-associated degradation (ERAD). Degradation of Pma1-D378N is dependent on the ubiquitin ligase Doa10 and the ubiquitin chaperone Cdc48. Recognition of Pma1-D378N by the ERAD pathway is dependent on Eps1, a transmembrane member of the protein disulfide isomerase (PDI) oxidoreductase family. Eps1 has two thioredoxin-like domains containing a CPHC and a CDKC active site. Although Eps1 interaction with wild-type Pma1 was not detected, Eps1 co-immunoprecipitates with Pma1-D378N. Eps1 interaction with Pma1-D378N requires the CPHC motif, although both thioredoxin-like domains appear to cooperate in substrate recognition. In the absence of the native transmembrane domain and cytoplasmic tail of Eps1, degradation of Pma1-D378N is slowed, suggesting that Eps1 facilitates presentation of substrate to membrane-bound components of the degradation machinery. Genetic interactions with other mutants of the ERAD machinery and induction of the unfolded protein response in eps1Delta cells support a general role for Eps1 as a recognition component of the ERAD pathway.     

The extracellular loops of Smoothened play a regulatory role in control of Hedgehog pathway activation

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.     

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p.

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.     

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.The endoplasmic reticulum (ER)² is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1, 2). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3, 4), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5, 6).In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7).The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man₉GlcNAc₂ oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9–11). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11–15), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14, 16–18). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1).After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20–23). The yeast PDI, Pdi1p, contains four thioredoxin-like domains, two of which have a CGHC motif as active sites, followed by a C-terminal extension containing the ER retention signal. During its catalytic cycle, PDI transiently forms a mixed disulfide intermediate with its substrate through an intermolecular disulfide bond between the cysteine residues of the active site of PDI and the substrate molecule.Here we report identification of PDI as an Mnl1p-interacting protein. Stable interactions between the C-terminal domain of Mnl1p and PDI involve intermolecular disulfide bonds. Stably interacting PDI is required for formation of the functionally essential intramolecular disulfide bond in the MHD of Mnl1p, which in turn stabilizes and prolongs the Mnl1p-PDI interactions. Possible roles for those stable interactions between Mnl1p and PDI in the ERAD will be discussed.     

Structural basis of an ERAD pathway mediated by the ER-resident protein disulfide reductase ERdj5

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.     

Suppression of Hedgehog signaling by Cul3 ligases in proliferation control of retinal precursors

Cullin-RING ubiquitin ligases ubiquitinate protein substrates and control their levels through degradation. Here we show that cullin3 (Cul3) suppresses Hedgehog (Hh) signaling through downregulating the level of the signaling pathway effector cubitus interruptus (Ci). High-level Hh signaling promotes Cul3-dependent Ci degradation, leading to the downregulation of Hh signaling. This process is manifested in controlling cell proliferation during Drosophila retinal development. In Cul3 mutants, the population of interommatidial cells is increased, which can be mimicked by overexpression of Ci and suppressed by depleting endogenous Ci. Hh also regulates the population of interommatidial cells in the pupal stage. Alterations in the interommatidial cell population correlate with alterations in precursor proliferation in the second mitotic wave of larval eye discs. Taken together, these results suggest that Cul3 downregulates Ci levels to modulate Hh signaling activity, thus ensuring proper cell proliferation during retinal development.     

Regulation of Mitochondrial Antiviral Signaling (MAVS) Expression and Signaling by the Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Protein Gp78

In a previous study, we identified the E3 ubiquitin ligase Gp78 by RNAi high-throughput screening as a gene whose depletion restricted enterovirus infection. In the current study, we show that Gp78, which localizes to the ER-mitochondria interface, is a regulator of RIG-I-like receptor (RLR) antiviral signaling. We show that depletion of Gp78 results in a robust decrease of vesicular stomatitis virus (VSV) infection and a corresponding enhancement of type I interferon (IFN) signaling. Mechanistically, we show that Gp78 modulates type I IFN induction by altering both the expression and signaling of the mitochondria-localized RLR adaptor mitochondrial antiviral signaling (MAVS). Expression of mutants of Gp78 that abolish its E3 ubiquitin ligase and its participation in ER-associated degradation (ERAD) lost their ability to degrade MAVS, but surprisingly maintained their ability to repress RLR signaling. In contrast, Gp78 lacking its entire C terminus lost both its ability to degrade MAVS and repress RLR signaling. We show that Gp78 interacts with both the N- and C-terminal domains of MAVS via its C-terminal RING domain, and that this interaction is required to abrogate Gp78-mediated attenuation of MAVS signaling. Our data thus implicate two parallel pathways by which Gp78 regulates MAVS signaling; one pathway requires its E3 ubiquitin ligase and ERAD activity to directly degrade MAVS, whereas the other pathway occurs independently of these activities, but requires the Gp78 RING domain and occurs via a direct association between this region and MAVS.     

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation.     

Disulfide structure of the leucine-rich repeat C-terminal cap and C-terminal stalk region of Nogo-66 receptor

Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.     

Glycosylation-independent ERAD pathway serves as a backup system under ER stress

During endoplasmic reticulum (ER)–associated degradation (ERAD), terminally misfolded proteins are retrotranslocated from the ER to the cytosol and degraded by the ubiquitin-proteasome system. Misfolded glycoproteins are recognized by calnexin and transferred to EDEM1, followed by the ER disulfide reductase ERdj5 and the BiP complex. The mechanisms involved in ERAD of nonglycoproteins, however, are poorly understood. Here we show that nonglycoprotein substrates are captured by BiP and then transferred to ERdj5 without going through the calnexin/EDEM1 pathway; after cleavage of disulfide bonds by ERdj5, the nonglycoproteins are transferred to the ERAD scaffold protein SEL1L by the aid of BiP for dislocation into the cytosol. When glucose trimming of the N-glycan groups of the substrates is inhibited, glycoproteins are also targeted to the nonglycoprotein ERAD pathway. These results indicate that two distinct pathways for ERAD of glycoproteins and nonglycoproteins exist in mammalian cells, and these pathways are interchangeable under ER stress conditions.     

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

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

Protein disulfide isomerases contribute differentially to the endoplasmic reticulum-associated degradation of apolipoprotein B and other substrates

ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.     

The human selenoprotein VCP-interacting membrane protein (VIMP) is non-globular and harbors a reductase function in an intrinsically disordered region

The human selenoprotein VIMP (VCP-interacting membrane protein)/SelS (selenoprotein S) localizes to the endoplasmic reticulum (ER) membrane and is involved in the process of ER-associated degradation (ERAD). To date, little is known about the presumed redox activity of VIMP, its structure and how these features might relate to the function of the protein in ERAD. Here, we use the recombinantly expressed cytosolic region of VIMP where the selenocysteine (Sec) in position 188 is replaced with a cysteine (a construct named cVIMP-Cys) to characterize redox and structural properties of the protein. We show that Cys-188 in cVIMP-Cys forms a disulfide bond with Cys-174, consistent with the presence of a Cys174-Sec188 selenosulfide bond in the native sequence. For the disulfide bond in cVIMP-Cys we determined the reduction potential to -200 mV, and showed it to be a good substrate of thioredoxin. Based on a biochemical and structural characterization of cVIMP-Cys using analytical gel filtration, CD and NMR spectroscopy in conjunction with bioinformatics, we propose a comprehensive overall structural model for the cytosolic region of VIMP. The data clearly indicate the N-terminal half to be comprised of two extended α-helices followed by a C-terminal region that is intrinsically disordered. Redox-dependent conformational changes in cVIMP-Cys were observed only in the vicinity of the two Cys residues. Overall, the redox properties observed for cVIMP-Cys are compatible with a function as a reductase, and we speculate that the plasticity of the intrinsically disordered C-terminal region allows the protein to access many different and structurally diverse substrates.     

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here, we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.     

Hedgehog activates the EGF receptor pathway during Drosophila head development.

The Hedgehog (Hh) and Epidermal growth factor receptor (EGFR) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, we demonstrate a direct link between these two pathways in Drosophila melanogaster. Hh and EGFR signaling are each required for the formation of a specific region of the head of the adult fruitfly. We show that hh and vein (vn), which encodes a ligand of the Drosophila EGFR (Schnepp, B., Grumbling, G., Donaldson, T. and Simcox, A. (1996) Genes Dev. 10, 2302-13), are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, we demonstrate that Hh activates vn expression. We also show that Hh activation of vn is mediated through the gene cubitus interruptus (ci) and that this activation requires the C-terminal region of the Ci protein. Finally, we demonstrate that wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling.