A first line of defense against ER stress

BiP is the predominant DnaK/Hsp70-type chaperone protein in the ER. It is required for folding and assembling newly synthesized ER client proteins, yet having too much BiP inhibits folding. In this issue, Chambers et al. (2012. J. Cell Biol. doi:10.1083/jcb.201202005) report that ADP ribosylation of BiP provides a reversible switch that fine tunes BiP activity according to need.     

Posttranslational folding of vesicular stomatitis virus G protein in the ER: involvement of noncovalent and covalent complexes

In this study, we show that posttranslational folding of Vesicular Stomatitis virus G protein subunits can involve noncovalent, multimeric complexes as transient intermediates. The complexes are heterogeneous in size (4-21S20,W), contain several G glycopolypeptides, and are associated with BiP/GRP78. The newly synthesized, partially intrachain disulfide-bonded G proteins enter these complexes immediately after chain termination, and are released 1-4 min later as fully oxidized, trimerization-competent monomers. These monomers are properly folded, judging by their binding of conformation-specific mAbs. When the G protein is translated in the presence of DTT, it remains reduced, largely unfolded and aggregated in the ER, but it can fold successfully when the DTT is removed. In this case, contrary to normal folding, the aggregates become transiently disulfide cross-linked. We also demonstrated that the fidelity of the folding process is dependent on metabolic energy. Finally, we established that the G protein of the folding mutant of the Vesicular Stomatitis virus, ts045, is blocked at a relatively late step in the folding pathway and remains associated with oligomeric, BiP/GRP78-containing folding complexes.     

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro

Parallel experiments in living cells and in vitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39 degrees C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32 degrees C, these properties were reversed, and the protein was transported to the cell surface. Upon the shift up from 32 degrees C back to 39 degrees C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.     

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.     

Arabidopsis thaliana has a set of J proteins in the endoplasmic reticulum that are conserved from yeast to animals and plants

J domain-containing proteins (J proteins) are functional partners for heat shock protein 70 (Hsp70) molecular chaperones and mediate various cellular processes by regulating activities of Hsp70. Budding yeast has three J proteins in the endoplasmic reticulum (ER): Scj1p and Jem1p functioning in protein folding and quality control in the ER, and Sec63p functioning in protein translocation across the ER membrane as partners for BiP, an Hsp70 in the ER. Here we report that Arabidopsis thaliana has orthologs of these yeast ER J proteins, which we designated as AtERdj3A, AtERdj3B, AtP58(IPK), AtERdj2A and AtERdj2B. Tunicamycin treatment of Arabidopsis cells, which causes ER stress, led to up-regulation of AtERdj3A, AtERdj3B, AtP58(IPK) and AtERdj2B. Subcellular fractionation analyses showed their ER localization, indicating that the identified J proteins indeed function as partners for BiP in Arabidopsis cells. Since expression of AtERdj3A, AtERdj3B and AtP58(IPK) partially suppressed the growth defects of the yeast jem1Deltascj1Delta mutant, they have functions similar to those of Scj1p and Jem1p. T-DNA insertions of the AtERDJ2A gene resulted in pollen germination defects, probably reflecting its essential function in protein translocation. These results suggest that A. thaliana has a set of ER J proteins structurally and functionally conserved from yeast to plants.     

Hepatic lipase maturation: a partial proteome of interacting factors

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.     

Saccharomyces cerevisiae Rot1p is an ER-localized membrane protein that may function with BiP/Kar2p in protein folding

The 70-kDa heat shock protein (Hsp70) family of molecular chaperones cooperates with cofactors to promote protein folding, assembly of protein complexes and translocation of proteins across membranes. Although many cofactors of cytosolic Hsp70s have been identified, knowledge about cofactors of BiP/Kar2p, an endoplasmic reticulum (ER)-resident Hsp70, is still poor. Here we propose the Saccharomyces cerevisiae protein Rot1p as a possible cofactor of BiP/Kar2p involved in protein folding. Rot1p was found to be an essential, ER-localized membrane protein facing the lumen. ROT1 genetically interacted with several ER chaperone genes including KAR2, and the rot1-2 mutation triggered the unfolded protein response. Rot1p associated with Kar2p, especially under conditions of ER stress, and maturation of a model protein, a reduced form of carboxypeptidaseY, was impaired in a kar2-1 rot1-2 double mutant. These findings suggest that Rot1p participates in protein folding with Kar2p. Morphological analysis of rot1-2 cells revealed cell wall defects and accumulation of autophagic bodies in the vacuole. This implies that the protein folding machinery in which Rot1p is involved chaperones proteins acting in various physiological processes including cell wall synthesis and lysis of autophagic bodies.     

Transient aggregation of nascent thyroglobulin in the endoplasmic reticulum: relationship to the molecular chaperone, BiP

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP-associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.     

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.     

BiP,an endoplasmic reticulum chaperone,modulates the development of morphine antinociceptive tolerance

Morphine is a potent analgesic, but the molecular mechanism for tolerance formation after repeated use is not fully understood. Binding immunoglobulin protein (BiP) is an endoplasmic reticulum (ER) chaperone that is central to ER function. We examined knock‐in mice expressing a mutant BiP with the retrieval sequence deleted in order to elucidate physiological processes that are sensitive to BiP functions. We tested the thermal antinociceptive effect of morphine in heterozygous mutant BiP mice in a hot plate test. Paw withdrawal latencies before and after a single administration of morphine were not significantly different between the wild‐type and mutant BiP mice. Repeated morphine administration caused the development of morphine tolerance in the wild‐type mice. The activation of glycogen synthase kinase 3b (GSK‐3b) was associated with morphine tolerance, because an inhibitor of GSK‐3β prevented it. On the other hand, the mutant BiP mice showed less morphine tolerance, and the activation of GSK‐3b was suppressed in their brain. These results suggest that BiP may play an important role in the development of morphine tolerance. Furthermore, we found that a chemical chaperone which improves ER protein folding capacity also attenuated the development of morphine tolerance in wild‐type mice, suggesting a possible clinical application of chemical chaperones in preventing morphine tolerance.     

ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.     

Transient Association of Calnexin and Calreticulin with Newly Synthesized G1 and G2 Glycoproteins of Uukuniemi Virus (Family Bunyaviridae)

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the Bunyaviridae family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257-266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.     

Cycloheximide, a new tool to dissect specific steps in ER-associated degradation of different substrates.

To study the degradation requirements of unassembled immunoglobulin (Ig) chains, we heterologously expressed a cDNA encoding the secretory form of murine mu in the yeast S. cerevisiae. We found that mu chains were translocated into and retained in the endoplasmic reticulum (ER) as they were N-glycosylated and bound to the yeast homolog of BiP, Kar2p. Similar to mutant yeast carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu protein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6p and Ubc7p or in cells overexpressing mutant ubiquitin. Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the accumulation of polyubiquitinated mu chains that were still glycosylated. By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step. In contrast to the situation for CPY*, the ER-transmembrane protein Der1p is not essential for mu degradation. Strikingly, however, the CHX-induced accumulation of polyubiquitinated Igmu chains was stronger in deltader1-mutants as compared to wild-type cells, indicating an additive effect of two inhibitory conditions. The results support a previously unknown activity of CHX, i.e. impairing the degradation of transport-incompetent secretory mu chains. Moreover, this activity will allow to dissect substrate-specific steps in ER associated protein degradation.     

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.     

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.     

Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species

Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport.     

The effect of calnexin deletion on the expression level of binding protein (BiP) under heat stress conditions in Saccharomyces cerevisiae

In order to investigate the effect of calnexin deletion on the induction of the main ER molecular chaperone BiP, we cultured the wild-type and calnexin-disrupted Saccharomyces cerevisiae strains under normal and stressed conditions. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced level of BiP mRNA in the ER was evidently higher in calnexin-disrupted S. cerevisiae than in the wild-type at 37°C, but was almost the same in the two strains under normal conditions. The Western blot analysis results for BiP protein expression in the ER showed a parallel in the mRNA levels in the two strains. It is suggested that under heat stress conditions, the induction of BiP in the ER might recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.     

Characterization and expression of the Douglas-fir luminal binding protein (PmBiP)

The endoplasmic reticulum (ER) molecular chaperone, BiP, plays a role in the cotranslational translocation and subsequent folding and assembly of newly synthesized proteins targeted to the ER and secretory pathway. The sequence encoding a Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) BiP homologue (PmBiP) was identified by differential screening of a seedling cDNA library. Southern blotting indicated that PmBiP is most likely present as a single copy. The deduced amino acid sequence of PmBiP contains an HEEL tetrapeptide sequence which functions to retain PmBiP in the ER and is different from HDEL commonly found in angiosperm plant BiPs. Amino acid sequence alignment and phylogenetic analysis show that PmBiP is highly similar to other plant BiPs yet forms a distinct phylogenetic subgroup which is separate from the angiosperm BiPs. Northern and western blotting revealed that PmBiP is subject to developmental regulation during seed development, germination, and early seedling growth and is seasonally regulated in needles of young seedlings. Received: 21 February 2000 / Accepted: 13 April 2000