Contribution of the HEDJ/ERdj3 cysteine-rich domain to substrate interactions

Cytoplasmic type I DnaJ/Hsp40 chaperones contain a Cys-rich domain consisting of four CXXCXG motifs that are in a reduced state and coordinate zinc, stabilizing the intervening sequence in a loop structure. However, the Cys-rich region of the endoplasmic reticulum localized HEDJ (ERdj3/ERj3p), is considerably different in sequence and arrangement. Unlike the typical type I molecule, the HEDJ CXC, and CXXC motifs were demonstrated in this study to be predominantly oxidized in intramolecular disulfide bonds. In the native state, HEDJ bound to immobilized, denatured thyroglobulin. Unlike its binding partner GRP78, redox conditions affected the interaction of HEDJ with substrate. Substitution of the Cys-rich domain cysteine residues with serine diminished or abolished HEDJ binding in the in vitro assay. These findings suggest that the Cys-rich region of HEDJ and its oxidation state are important in maintaining the substrate interaction domain in a binding-competent conformation.     

Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress

The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His --> Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation.     

Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions

Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Deltaerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Deltascj1Deltajem1) and in the induction of the unfolded protein response (Deltaire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.     

An unusual TOM20/TOM22 bypass mechanism for the mitochondrial targeting of cytochrome P450 proteins containing N-terminal chimeric signals

Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP+33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP+5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondria-targeting signals are highly evolved and dynamic in nature.     

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.     

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.     

Steered molecular dynamics simulation of the binding of the bovine auxilin J domain to the Hsc70 nucleotide-binding domain

The Hsp70 and Hsp40 chaperone machine plays critical roles in protein folding, membrane translocation, and protein degradation by binding and releasing protein substrates in a process that utilizes ATP. The activities of the Hsp70 family of chaperones are recruited and stimulated by the J domains of Hsp40 chaperones. However, structural information on the Hsp40–Hsp70 complex is lacking, and the molecular details of this interaction are yet to be elucidated. Here we used steered molecular dynamics (SMD) simulations to investigate the molecular interactions that occur during the dissociation of the auxilin J domain from the Hsc70 nucleotide-binding domain (NBD). The changes in energy observed during the SMD simulation suggest that electrostatic interactions are the dominant type of interaction. Additionally, we found that Hsp70 mainly interacts with auxilin through the surface residues Tyr866, Arg867, and Lys868 of helix II, His874, Asp876, Lys877, Thr879, and Gln881 of the HPD loop, and Phe891, Asn895, Asp896, and Asn903 of helix III. The conservative residues Tyr866, Arg867, Lys868, His874, Asp876, Lys877, and Phe891 were also found in a previous study to be indispensable to the catalytic activity of the DnaJ J domain and the binding of it with the NBD of DnaK. The in silico identification of the importance of auxilin residues Asn895, Asp896, and Asn903 agrees with previous mutagenesis and NMR data suggesting that helix III of the J domain of the T antigen interacts with Hsp70. Furthermore, our data indicate that Thr879 and Gln881 from the HPD loop are also important as they mediate the interaction between the bovine auxilin J domain and Hsc70.     

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.     

Causal Links Between Protein Folding in the ER and Events Along the Secretory Pathway

The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes.

Addendum to:

Saccharomyces cerevisiae Rot1p is an ER-Localized Membrane Protein that May Function with BiP/Kar2p in Protein Folding

Masato Takeuchi, Yukio Kimata, Aiko Hirata, Masahiro Oka and Kenji Kohno

J Biochem 2006; 139:597-605     

Overexpression of yeast Hsp110 homolog Sse1p suppresses ydj1-151 thermosensitivity and restores Hsp90-dependent activity

The Saccharomyces cerevisiae heat-shock protein (Hsp)40, Ydj1p, is involved in a variety of cellular activities that control polypeptide fate, such as folding and translocation across intracellular membranes. To elucidate the mechanism of Ydj1p action, and to identify functional partners, we screened for multicopy suppressors of the temperature-sensitive ydj1-151 mutant and identified a yeast Hsp110, SSE1. Overexpression of Sse1p also suppressed the folding defect of v-Src kinase in the ydj1-151 mutant and partially reversed the alpha-factor translocation defect. SSE1-dependent suppression of ydj1-151 thermosensitivity required the wild-type ATP-binding domain of Sse1p. However, the Sse1p mutants maintained heat-denatured firefly luciferase in a folding-competent state in vitro and restored human androgen receptor folding in sse1 mutant cells. Because the folding of both v-Src kinase and human androgen receptor in yeast requires the Hsp90 complex, these data suggest that Ydj1p and Sse1p are interacting cochaperones in the Hsp90 complex and facilitate Hsp90-dependent activity.     

Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex.

Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes. We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane. In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis. In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied. An intact peptide binding pocket is required for this interaction. Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves.     

BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane.

We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.     

Causal links between protein folding in the ER and events along the secretory pathway

The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes.     

Molecular characterization and expression of a gene encoding cytosolic Hsp90 from Pennisetum glaucum and its role in abiotic stress adaptation

Heat shock protein 90 (Hsp90) is an abundant and highly conserved molecular chaperone that is essential for viability in eukaryotes. They have a crucial role in the folding of a set of proteins involved in the regulation of many essential cellular pathways and also re-folding of stress-denatured polypeptides. However, their exact function is still not clearly elucidated. In this study the full-length cDNA encoding for Hsp90 polypeptide and its corresponding gene was isolated from Pennisetum glaucum (designated PgHsp90). PgHsp90 cDNA encoded for a polypeptide of 698 amino acids with a predicted molecular mass of 80.3kDa and shared a high sequence homology (97-81%) to other plant cytosolic Hsp90s and shared less sequence homology (40-45%) to organelle and endoplasmic reticulum specific Hsp90 isoforms. A deduced amino acid sequence possessed three structural domains: N-terminus (1-211) ATP binding domain, middle (281-540) client protein interacting domain and C-terminus (541-698) dimerization domain; the N-terminus and middle domain is linked by a charged linker domain (212-280). It possesses the five-conserved amino acid signature sequence motifs characteristic of the Hsp90 family and a C-terminus MEEVD penta-peptide characteristic of the cytosolic Hsp90 isoform. The predicted quaternary architecture generated for PgHsp90 through molecular modeling was globally akin to that of yeast Hsp90. The PgHsp90 gene consists of 3 exons and 2 introns. The position and phasing of these introns were conserved in other plant cytosolic Hsp90 genes. Recombinant PgHsp90 protein was expressed in E. coli and purified to homogeneity, which possessed in vitro chaperone activity. E. coli expressing PgHsp90 protein showed enhanced tolerance to heat, salt and dehydration stresses. The quantitative up-regulation of PgHsp90 gene expression positively correlates in response to different stresses to meet the additional demand for protein folding support. Cumulatively, the in vivo and in vitro experiments indicated that PgHsp90 plays an adaptive or protective role to counter the stress induced protein damage.     

Heat shock proteins as molecular chaperones

Exposure to different conditions or agents that destabilize cell homeostasis often alters protein folding. Depending on stress intensity irreversible protein aggregation and cell death can occur. Cells have developed a conserved defense mechanism aimed at reducing the deleterious effects induced by protein folding alteration. This mechanism is characterized by the expression of a small number of genes encoding specific proteins, named Hsps. Several of these proteins act as molecular chaperones through their ability to refold polypeptides with an altered conformation. Moreover, constitutive Hsps homologues have been characterized that participate in the folding of newly made polypeptides, in the assembly of protein complexes in the endoplasmic reticulum, in the translocation of polypeptides through membranes or in masking mutations that alter protein folding. Neurodegeneratives and cancereous diseases are discussed as examples where high levels of Hsp expression can be either beneficial or deleterious to the cells.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.     

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.