A J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.

An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP.

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria.

In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the "posttranslational" translocation pathway.

Ribosome-associated chaperones as key players in proteostasis.

De novo protein folding is delicate and error-prone and requires the guidance of molecular chaperones. Besides cytosolic and organelle-specific chaperones, cells have evolved ribosome-associated chaperones that support early folding events and prevent misfolding and aggregation. This class of chaperones includes the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-associated complex (NAC) and specialized eukaryotic heat shock protein (Hsp) 70/40 chaperones. This review focuses on the cellular activities of ribosome-associated chaperones and highlights new findings indicating additional functions beyond de novo folding. These activities include the assembly of oligomeric complexes, such as ribosomes, modulation of translation and targeting of proteins.

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.

The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome.

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.

Structural analysis of the ribosome-associated complex (RAC) reveals an unusual Hsp70/Hsp40 interaction.

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.

Directed PCR-free engineering of highly repetitive DNA sequences.

BACKGROUND: Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. RESULTS: For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. CONCLUSION: Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD.

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.

Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes.

The metazoan endoplasmic reticulum (ER) serves both as a hub for maturation of secreted proteins and as an intracellular calcium storage compartment, facilitating calcium-release-dependent cellular processes. ER calcium depletion robustly activates the unfolded protein response (UPR). However, it is unclear how fluctuations in ER calcium impact organellar proteostasis. Here, we report that calcium selectively affects the dynamics of the abundant metazoan ER Hsp70 chaperone BiP, by enhancing its affinity for ADP. In the calcium-replete ER, ADP rebinding to post-ATP hydrolysis BiP-substrate complexes competes with ATP binding during both spontaneous and co-chaperone-assisted nucleotide exchange, favouring substrate retention. Conversely, in the calcium-depleted ER, relative acceleration of ADP-to-ATP exchange favours substrate release. These findings explain the rapid dissociation of certain substrates from BiP observed in the calcium-depleted ER and suggest a mechanism for tuning ER quality control and coupling UPR activity to signals that mobilise ER calcium in secretory cells.

Early Events in the Endoplasmic Reticulum Unfolded Protein Response.

The physiological consequences of the unfolded protein response (UPR) are mediated by changes in gene expression. Underlying them are rapid processes involving preexisting components. We review recent insights gained into the regulation of the endoplasmic reticulum (ER) Hsp70 chaperone BiP, whose incorporation into inactive oligomers and reversible AMPylation and de-AMPylation present a first line of response to fluctuating levels of unfolded proteins. BiP activity is tied to the regulation of the UPR transducers by a recently discovered cycle of ER-localized, J protein-mediated formation of a repressive IRE1-BiP complex, whose working we contrast to an alternative model for UPR regulation that relies on direct recognition of unfolded proteins. We conclude with a discussion of mechanisms that repress messenger RNA (mRNA) translation to limit the flux of newly synthesized proteins into the ER, a rapid adaptation that does not rely on new macromolecule biosynthesis.

MANF antagonizes nucleotide exchange by the endoplasmic reticulum chaperone BiP.

Despite its known role as a secreted neuroprotectant, much of the mesencephalic astrocyte-derived neurotrophic factor (MANF) is retained in the endoplasmic reticulum (ER) of producer cells. There, by unknown mechanisms, MANF plays a role in protein folding homeostasis in complex with the ER-localized Hsp70 chaperone BiP. Here we report that the SAF-A/B, Acinus, and PIAS (SAP) domain of MANF selectively associates with the nucleotide binding domain (NBD) of ADP-bound BiP. In crystal structures the SAP domain engages the cleft between NBD subdomains Ia and IIa, stabilizing the ADP-bound conformation and clashing with the interdomain linker that occupies this site in ATP-bound BiP. MANF inhibits both ADP release from BiP and ATP binding to BiP, and thereby client release. Cells lacking MANF have fewer ER stress-induced BiP-containing high molecular weight complexes. These findings suggest that MANF contributes to protein folding homeostasis as a nucleotide exchange inhibitor that stabilizes certain BiP-client complexes.

FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.

Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD-/- hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP contributes to protein folding homeostasis by engaging unfolded client proteins in a process that is tightly coupled to ATP binding and hydrolysis. The inverse correlation between BiP AMPylation and the burden of unfolded ER proteins suggests a post-translational mechanism for adjusting BiP's activity to changing levels of ER stress, but the underlying molecular details are unexplored. We present biochemical and crystallographic studies indicating that irrespective of the identity of the bound nucleotide AMPylation biases BiP towards a conformation normally attained by the ATP-bound chaperone. AMPylation does not affect the interaction between BiP and J-protein co-factors but appears to allosterically impair J protein-stimulated ATP-hydrolysis, resulting in the inability of modified BiP to attain high affinity for its substrates. These findings suggest a molecular mechanism by which AMPylation serves as a switch to inactivate BiP, limiting its interactions with substrates whilst conserving ATP.

AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker.

DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP. Native gel electrophoresis revealed a rapidly-modulated reciprocal relationship between the burden of unfolded proteins and BiP oligomers and slower equilibration between oligomers and inactive, covalently-modified BiP. Lumenal ER calcium depletion caused rapid oligomerization of mammalian BiP and a coincidental diminution in substrate binding, pointing to the relative inertness of the oligomers. Thus, equilibration between inactive oligomers and active monomeric BiP is poised to buffer fluctuations in ER unfolded protein load on a rapid timescale attainable neither by inter-conversion of active and covalently-modified BiP nor by the conventional unfolded protein response.

A dual function for chaperones SSB-RAC and the NAC nascent polypeptide-associated complex on ribosomes.

The yeast Hsp70/40 system SSB-RAC (stress 70 B-ribosome-associated complex) binds to ribosomes and contacts nascent polypeptides to assist cotranslational folding. In this study, we demonstrate that nascent polypeptide-associated complex (NAC), another ribosome-tethered system, is functionally connected to SSB-RAC and the cytosolic Hsp70 network. Simultaneous deletions of genes encoding NAC and SSB caused conditional loss of cell viability under protein-folding stress conditions. Furthermore, NAC mutations revealed genetic interaction with a deletion of Sse1, a nucleotide exchange factor regulating the cytosolic Hsp70 network. Cells lacking SSB or Sse1 showed protein aggregation, which is enhanced by additional loss of NAC; however, these mutants differ in their potential client repertoire. Aggregation of ribosomal proteins and biogenesis factors accompanied by a pronounced deficiency in ribosomal particles and translating ribosomes only occurs in ssbDelta and nacDeltassbDelta cells, suggesting that SSB and NAC control ribosome biogenesis. Thus, SSB-RAC and NAC assist protein folding and likewise have important functions for regulation of ribosome levels. These findings emphasize the concept that ribosome production is coordinated with the protein-folding capacity of ribosome-associated chaperones.