The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum - a quantitative approach

Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell-free studies on protein transport into the mammalian ER. These studies have revealed multiple roles for the major ER-luminal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at least one of which also involves the second ER-luminal Hsp70, glucose-regulated protein (Grp) 170. In addition, at least one of these BiP activities depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types. Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes. We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP. The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER-membrane proteins ER-resident J-domain protein 1 plus ER-resident J-domain protein 2/Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER. Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and Marinesco-Sj?gren syndrome.     

Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.     

LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum

Lhs1p is an Hsp70-related chaperone localized in the endoplasmic reticulum (ER) lumen. Deltalhs1 mutant cells are viable but are constitutively induced for the unfolded protein response (UPR). Here, we demonstrate a severe growth defect in Deltaire1Deltalhs1 double mutant cells in which the UPR can no longer be induced. In addition, we have identified a UPR- regulated gene, SIL1, whose overexpression is sufficient to suppress the Deltaire1Deltalhs1 growth defect. SIL1 encodes an ER-localized protein that interacts directly with the ATPase domain of Kar2p (BiP), suggesting some role in modulating the activity of this vital chaperone. SIL1 is a non-essential gene but the Deltalhs1Deltasil1 double mutation is lethal and correlates with a complete block of protein translocation into the ER. We conclude that the IRE1-dependent induction of SIL1 is a vital adaptation in Deltalhs1 cells, and that the activities associated with the Lhs1 and Sil1 proteins constitute an essential function required for protein translocation into the ER. The Sil1 protein appears widespread amongst eukaryotes, with homologues in Yarrowia lipolytica (Sls1p), Drosophila and mammals.     

The Lhs1/GRP170 Chaperones Facilitate the Endoplasmic Reticulum-associated Degradation of the Epithelial Sodium Channel

The epithelial sodium channel, ENaC, plays a critical role in maintaining salt and water homeostasis, and not surprisingly defects in ENaC function are associated with disease. Like many other membrane-spanning proteins, this trimeric protein complex folds and assembles inefficiently in the endoplasmic reticulum (ER), which results in a substantial percentage of the channel being targeted for ER-associated degradation (ERAD). Because the spectrum of factors that facilitates the degradation of ENaC is incomplete, we developed yeast expression systems for each ENaC subunit. We discovered that a conserved Hsp70-like chaperone, Lhs1, is required for maximal turnover of the ENaC α subunit. By expressing Lhs1 ATP binding mutants, we also found that the nucleotide exchange properties of this chaperone are dispensable for ENaC degradation. Consistent with the precipitation of an Lhs1-αENaC complex, Lhs1 holdase activity was instead most likely required to support the ERAD of αENaC. Moreover, a complex containing the mammalian Lhs1 homolog GRP170 and αENaC co-precipitated, and GRP170 also facilitated ENaC degradation in human, HEK293 cells, and in a Xenopus oocyte expression system. In both yeast and higher cell types, the effect of Lhs1 on the ERAD of αENaC was selective for the unglycosylated form of the protein. These data establish the first evidence that Lhs1/Grp170 chaperones can act as mediators of ERAD substrate selection.     

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.     

Interactions between Kar2p and Its Nucleotide Exchange Factors Sil1p and Lhs1p Are Mechanistically Distinct

Kar2p, an essential Hsp70 chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae, facilitates the transport and folding of nascent polypeptides within the endoplasmic reticulum lumen. The chaperone activity of Kar2p is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, namely Sil1p and Lhs1p. Here, we demonstrate that the binding requirements for Lhs1p are complex, requiring both the nucleotide binding domain plus the linker domain of Kar2p. In contrast, the IIB domain of Kar2p is sufficient for binding of Sil1p, and point mutations within IIB specifically blocked Sil1p-dependent activation while remaining competent for activation by Lhs1p. Taken together, these results demonstrate that the interactions between Kar2p and its two nucleotide exchange factors can be functionally resolved and are thus mechanistically distinct.     

The Grp170 nucleotide exchange factor executes a key role during ERAD of cellular misfolded clients

When a protein misfolds in the endoplasmic reticulum (ER), it retrotranslocates to the cytosol and is degraded by the proteasome via a pathway called ER-associated degradation (ERAD). To initiate ERAD, ADP-BiP is often recruited to the misfolded client, rendering it soluble and translocation competent. How the misfolded client is subsequently released from BiP so that it undergoes retrotranslocation, however, remains enigmatic. Here we demonstrate that the ER-resident nucleotide exchange factor (NEF) Grp170 plays an important role during ERAD of the misfolded glycosylated client null Hong Kong (NHK). As a NEF, Grp170 triggers nucleotide exchange of BiP to generate ATP-BiP. ATP-BiP disengages from NHK, enabling it to retrotranslocate to the cytosol. We demonstrate that Grp170 binds to Sel1L, an adapter of the transmembrane Hrd1 E3 ubiquitin ligase postulated to be the retrotranslocon, and links this interaction to Grp170’s function during ERAD. More broadly, Grp170 also promotes degradation of the nonglycosylated transthyretin (TTR) D18G misfolded client. Our findings thus establish a general function of Grp170 during ERAD and suggest that positioning this client-release factor at the retrotranslocation site may afford a mechanism to couple client release from BiP and retrotranslocation.     

C-terminal mutations destabilize SIL1/BAP and can cause Marinesco-Sjögren syndrome

Marinesco-Sjögren syndrome (MSS) is an autosomal recessive, neurodegenerative, multisystem disorder characterized by severe phenotypes developing in infancy. Recently, mutations in the endoplasmic reticulum (ER)-associated co-chaperone SIL1/BAP were identified to be the major cause of MSS. SIL1 acts as a nucleotide exchange factor for BiP, the ER Hsp70 orthologue, which plays an essential role in the folding and assembly of nascent polypeptide chains in the ER. SIL1 facilitates the release of BiP from unfolded protein substrates, enabling the subsequent folding and transport of the protein. Although most mutations leading to MSS result in deletion of the majority of the protein, three separate mutations have been identified that disrupt only the last five or six amino acids of the protein, which were assumed to encode a divergent ER retention motif. This study presents an in depth analysis of two of these mutants and reveals that the phenotype in the affected individuals is not likely to be due to depletion of SIL1 from the ER via secretion. Instead, our analyses show that the mutant proteins are particularly unstable and either form large aggregates in the ER or are rapidly degraded via the proteasome. In agreement with our findings, homology modeling suggests that the very C-terminal residues of SIL1 play a role in its structural integrity rather than its localization. These new insights might be a first step toward a possible pharmacological treatment of certain types of MSS by specifically stabilizing the mutant SIL1 protein.     

BAP,a mammalian BiP-associated protein,is a nucleotide exchange factor that regulates the ATPase activity of BiP

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.     

The nucleotide exchange factors Grp170 and Sil1 induce cholera toxin release from BiP to enable retrotranslocation

Cholera toxin (CT) intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER), where the catalytic CTA1 subunit hijacks components of the ER-associated degradation (ERAD) machinery to retrotranslocate to the cytosol and induce toxicity. In the ER, CT targets to the ERAD machinery composed of the E3 ubiquitin ligase Hrd1-Sel1L complex, in part via the activity of the Sel1L-binding partner ERdj5. This J protein stimulates BiP''s ATPase activity, allowing BiP to capture the toxin. Presumably, toxin release from BiP must occur before retrotranslocation. Here, using loss-and gain-of-function approaches coupled with binding studies, we demonstrate that the ER-resident nucleotide exchange factors (NEFs) Grp170 and Sil1 induce CT release from BiP in order to promote toxin retrotranslocation. In addition, we find that after NEF-dependent release from BiP, the toxin is transferred to protein disulfide isomerase; this ER redox chaperone is known to unfold CTA1, which allows the toxin to cross the Hrd1-Sel1L complex. Our data thus identify two NEFs that trigger toxin release from BiP to enable successful retrotranslocation and clarify the fate of the toxin after it disengages from BiP.     

The Large Hsp70 Grp170 Binds to Unfolded Protein Substrates in Vivo with a Regulation Distinct from Conventional Hsp70s

The Hsp70 superfamily is a ubiquitous chaperone class that includes conventional and large Hsp70s. BiP is the only conventional Hsp70 in the endoplasmic reticulum (ER) whose functions include: assisting protein folding, targeting misfolded proteins for degradation, and regulating the transducers of the unfolded protein response. The ER also possesses a single large Hsp70, the glucose-regulated protein of 170 kDa (Grp170). Like BiP it is an essential protein, but its cellular functions are not well understood. Here we show that Grp170 can bind directly to a variety of incompletely folded protein substrates in the ER, and as expected for a bona fide chaperone, it does not interact with folded secretory proteins. Our data demonstrate that Grp170 and BiP associate with similar molecular forms of two substrate proteins, but while BiP is released from unfolded substrates in the presence of ATP, Grp170 remains bound. In comparison to conventional Hsp70s, the large Hsp70s possess two unique structural features: an extended C-terminal α-helical domain and an unstructured loop in the putative substrate binding domain with an unknown function. We find that in the absence of the α-helical domain the interaction of Grp170 with substrates is reduced. In striking contrast, deletion of the unstructured loop results in increased binding to substrates, suggesting the presence of unique intramolecular mechanisms of control for the chaperone functions of large Hsp70s.     

PERK inhibition attenuates the abnormalities of the secretory pathway and the increased apoptotic rate induced by SIL1 knockdown in HeLa cells

Loss-of-function mutations in the SIL1 gene are linked to Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of infancy characterized by cerebellar and skeletal muscle degeneration. SIL1 is a ubiquitous adenine nucleotide exchange factor for the endoplasmic reticulum (ER) chaperone BiP. The complexity of mechanisms by which loss of SIL1 causes MSS is not yet fully understood. We used HeLa cells to test the hypothesis that impaired protein folding in the ER due to loss of SIL1 could affect secretory trafficking, impairing the transport of cargoes essential for the function of MSS vulnerable cells. Immunofluorescence and ultrastructural analysis of SIL1-knocked-down cells detected ER chaperone aggregation, enlargement of the Golgi complex, increased autophagic vacuoles, and mitochondrial swelling. SIL1-interefered cells also had delayed ER-to-plasma membrane transport with retention of Na⁺/K⁺-ATPase and procollagen-I in the ER and Golgi, and increased apoptosis. The PERK pathway of the unfolded protein response was activated in SIL1-interfered cells, and the PERK inhibitor GSK2606414 attenuated the morphological and functional alterations of the secretory pathway, and significantly reduced cell death. These results indicate that loss of SIL1 is associated with alterations of secretory transport, and suggest that inhibiting PERK signalling may alleviate the cellular pathology of SIL1-related MSS.     

ER chaperones in mammalian development and human diseases

The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co-chaperones SIL1 and P58(IPK) and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.     

Sil1, a nucleotide exchange factor for BiP,is not required for antibody assembly or secretion

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco–Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1^Gt mouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1^Gt mouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1^Gt mice. ER chaperones were expressed at the same level in Sil1^WT and Sil1^Gt mice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production.     

Protein transport into the endoplasmic reticulum: mechanisms and pathologies

Transport into the endoplasmic reticulum (ER) is the crucial step in the biosynthesis of most secretory proteins and many membrane proteins. The products of the SIL1, SEC62 and SEC63 genes act in concert with the SEC61 complex and the molecular chaperones BiP and GRP170 to transport proteins into the ER. Interestingly, recent genetic work has linked mutations in the human and murine SIL1 genes to neurodegeneration, and mutations in the human SEC63 gene to autosomal dominant polycystic liver disease. Furthermore, mutations in the SEC63 gene and overexpression of the SEC62 gene are associated with various human cancers. Therefore, we suggest that these diseases should be considered to be pathologies of protein transport into the ER rather than protein-folding diseases.     

Androgens modulate autophagy and cell death via regulation of the endoplasmic reticulum chaperone glucose-regulated protein 78/BiP in prostate cancer cells

Pro-survival signalling mediated by the androgen receptor (AR) is implicated as a key contributor to prostate carcinogenesis. As prostate tumours are characterized by nutrient-poor, hypoxic and acidified microenvironments, one mechanism whereby AR signalling may contribute to survival is by promoting adaptation to cellular stress. Here we have identified a novel role for AR in the inhibition of autophagy induced by serum withdrawal. This blockade is attributed to AR-mediated upregulation of the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78/BiP (Grp78/BiP), and occurs independently of ER stress response pathway activation. Interestingly, AR activation did not affect serum starvation-induced mammalian target of rapamycin inhibition, illustrating that the adaptive role for androgens lies not in the ability to modulate nutrient sensing, but in the promotion of ER stability. Finally, we show that the adaptive advantage conferred by AR-mediated Grp78/BiP upregulation is temporary, as upon chronic serum starvation, AR activation delayed but did not suppress the onset of autophagy and cell death. This study reveals a novel mechanism whereby maintained AR signalling promotes temporary adaptation to cellular stress and in turn may contribute to the evasion of prostate tumour cell death.     

The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.     

Activation of mammalian IRE1α upon ER stress depends on dissociation of BiP rather than on direct interaction with unfolded proteins

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response. Upon ER stress, IRE1 senses the accumulation of unfolded proteins in the ER, and transfers signal from the ER to the cytosol. Recently, it was reported that the luminal domain of yeast Ire1 senses the unfolded proteins via a two-step mechanism, namely dissociation of BiP and direct interaction with unfolded proteins. However, it has been unclear whether a similar mechanism is applicable to mammalian IRE1α. To address this point, we analyzed luminal-domain mutants of mammalian IRE1α in cells, and evaluated the anti-aggregation activity of the luminal fragment of IRE1α in vitro. We generated a mutant that has low affinity for BiP, and this mutant was significantly activated even under normal conditions. Moreover, the luminal fragments of mammalian IRE1α did not exhibit anti-aggregation activity. These results suggest that in contrast to yeast Ire1, the regulation of mammalian IRE1α strongly depends on the dissociation of BiP.