The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum

Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin–proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated.     

The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system

The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import-defective mutated derivatives of carboxypeptidase yscY (DeltassCPY* and DeltassCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (DeltassCPY). All these protein species are rapidly degraded via the ubiquitin-proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssa1p function. Fusion of DeltassCPY* to GFP-cODC to form DeltassCPY*-GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation

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Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3

P58/DNAJc3 defends cells against endoplasmic reticulum (ER) stress. Most P58 molecules are translocated into the ER lumen, and here we report selective and stable binding to misfolded proteins by P58's TPR-containing N-terminal domain. In vitro, too, P58 binds selectively to a model misfolded protein and challenge of that complex with physiological concentrations of the ER lumenal Hsp70-type chaperone BiP encourages disassembly. BiP-induced dissociation of P58 from its substrate depends on the presence of ATP and on interactions with P58's J-domain, which are mediated by invariant residues BiP(R197) and P58(H422). A functional J-domain also accelerates dissociation of P58 from a model substrate, VSV-G(ts045), on the latter's re-folding in vivo. However, J-domain binding can be separated from the ability to promote substrate dissociation by the mutant BiP(E201G) and a wild-type J-domain fused ectopically to P58(H422Q) rescues the latter's inability to dissociate from substrate in response to BiP and ATP. These findings are consistent with a model whereby localized activation of the Hsp70-type partner is sufficient to promote substrate handover from the J-domain co-chaperone.     

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.     

Retention of membrane proteins by the endoplasmic reticulum

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope- specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi- associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.     

Topogenesis of membrane proteins at the endoplasmic reticulum

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences, which induce the translocation of either their N- or their C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated N-terminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro cross-linking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEbeta translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein.     

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

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

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.     

A class of membrane proteins shaping the tubular endoplasmic reticulum

How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/NogoA, a member of the ubiquitous reticulon family. Both in yeast and mammalian cells, the reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1p, a conserved integral membrane protein that also localizes to the tubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these "morphogenic" proteins partition into and stabilize highly curved ER membrane tubules.     

Integration of membrane proteins into the endoplasmic reticulum requires GTP

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment.

The use of two-dimensional gel electrophoresis has identified the gene products A14L (p16) and A13L (p8) as abundant membrane proteins of the first infectious form of vaccinia virus, the intracellular mature virus (IMV; O. N. Jensen, T. Houthaeve, A. Shevchenko, S. Cudmore, T. Ashford, M. Mann, G. Griffiths, J. Krijnse Locker, J. Virol. 70:7485-7497, 1996). In this study, these two proteins were characterized in detail. In infected cells, both proteins localize not only to the viral membranes but also to tubular-cisternal membranes of the intermediate compartment, defined by the use of antibodies to either rab1A or p21, which colocalize with rab1A (J. Krijnse Locker, S. Schleich, D. Rodriguez, B. Goud, E. J. Snijder, and G. Griffiths, J. Biol. Chem. 271:14950-14958, 1996). Both proteins appear to reach this destination via cotranslational insertion into the rough endoplasmic reticulum, as shown by in vitro translation and translocation experiments. Whereas p16 probably spans the membrane twice, p8 is inserted into the membrane by means of its single NH2-terminal hydrophobic domain, adopting a topology which leaves the C terminus exposed to the cytoplasm. Combined immunocytochemical and biochemical data show that p16 is a member of the inner of the two IMV membrane layers, whereas p8 localizes to both the inner and the outer membrane. These findings are discussed with respect to our model of IMV membrane assembly.     

Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.     

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.     

The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress

DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner.