Hsp70 Nucleotide Exchange Factor Research Articles

Escherichia coli-based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756cells are unable to support λ bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756cells grow adequately at temperatures ranging from 30 °C to 37 °C, but they die at elevated temperatures (>40 °C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Overexpression of the OsFes1A increased the phytosterols content and enhanced drought and salt stress tolerance in Arabidopsis.

Overexpression of the rice gene, OsFes1A, increased phytosterol content and drought and salt stress tolerance in Arabidopsis. Phytosterols are key components of the phospholipid bilayer membrane and regulate various processes of plant growth and response to biotic and abiotic stresses. In this study, it was demonstrated that the overexpression of OsFes1A (Hsp70 nucleotide exchange factor Fes1) increased phytosterols content and enhanced tolerance to salt and drought stress in Arabidopsis. In transgenic plants, the average content of campesterol was 17.6% higher than that of WT, and the average content of β-sitosterol reached 923.75μg/g, with an increase of 1.33-fold. In fes1a seeds, the contents of campesterol and β-sitosterol reduced by 20% and 10.93%, respectively. In OsFes1A transgenic seeds, the contents of campesterol and β-sitosterol increased by 1.38-fold and 1.25-fold respectively. Furthermore, the germination rate of transgenic Arabidopsis was significantly higher than WT under stress (salt, ABA, and drought treatment). Under salt stress, transgenic plants accumulated a lower MDA content, higher chlorophyll content, and POD activity relative to the wild type, while the mutants showed the opposite pattern Our study found multiple other functions of OsFes1A beyond the defined role of Fes1 in regulating Hsp70, contributing to the better understanding of the essential roles of Fes1 in plants. Meanwhile, it provides the theoretical basis for developing high phytosterol crop varieties.

Suppression of aggregate and amyloid formation by a novel intrinsically disordered region in metazoan Hsp110 chaperones

Molecular chaperones maintain proteostasis by ensuring the proper folding of polypeptides. Loss of proteostasis has been linked to numerous neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's disease. Hsp110 is related to the canonical Hsp70 class of protein-folding molecular chaperones and interacts with Hsp70 as a nucleotide exchange factor (NEF). In addition to its NEF activity, Hsp110 possesses an Hsp70-like substrate-binding domain (SBD) whose biological roles remain undefined. Previous work in Drosophila melanogaster has implicated the sole Hsp110 gene (Hsc70cb) in proteinopathic neurodegeneration. We hypothesize that in addition to its role as an Hsp70 NEF, Drosophila Hsp110 may function as a protective protein "holdase," preventing the aggregation of unfolded polypeptides via the SBD-β subdomain. We demonstrate for the first time that Drosophila Hsp110 effectively prevents aggregation of the model substrate citrate synthase. We also report the discovery of a redundant and heretofore unknown potent holdase capacity in a 138-amino-acid region of Hsp110 carboxyl terminal to both SBD-β and SBD-α (henceforth called the C-terminal extension). This sequence is highly conserved in metazoan Hsp110 genes, completely absent from fungal representatives, and is computationally predicted to contain an intrinsically disordered region (IDR). We demonstrate that this IDR sequence within the human Hsp110s, Apg-1 and Hsp105α, inhibits the formation of amyloid Aβ-42 and α-synuclein fibrils in vitro but cannot mediate fibril disassembly. Together these findings establish capacity for metazoan Hsp110 chaperones to suppress both general protein aggregation and amyloidogenesis, raising the possibility of exploitation of this IDR for therapeutic benefit.

Hsp70-nucleotide exchange factor (NEF) Fes1 has non-NEF roles in degradation of gluconeogenic enzymes and cell wall integrity.

Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles.

Methionine sulfoxide reductase reverses oxidation of the nucleotide exchange factor Fes1 to regulate cytoplasmic Hsp70 chaperone cycle

Methionine residues in proteins are targets of oxidation by reactive oxygen species (ROS). Selective oxidation of methionine to methionine sulfoxide (MetO) can allow for reversible, beneficial modification of proteins to limit cell damage during conditions of excess ROS. Methionine sulfoxide reductases (Msrs) are conserved enzymes that reduce MetO residues in proteins, serving to both limit the over‐oxidation of protein methionines, and to remove MetO adducts when stress subsides. Although loss of Msr function has been implicated in neurodegenerative disorders and aging, it is unclear how Msr activities influence these disease pathologies because the vast majority of their physiological substrates remain unknown.We have identified the cytoplasmic Hsp70 nucleotide exchange factor Fes1 as an Msr substrate in yeast. Under oxidative stress, Fes1 is oxidized at a cluster of N‐terminal methionine residues. Current data show that this Msr‐controlled modification reversibly regulates interaction between Fes1 and Hsp70, thereby modulating chaperone nucleotide exchange, ATP hydrolysis, and peptide binding and release. We suggest that these altered Hsp70 activities, as a consequence of Fes1 modification, may help cells cope with elevated ROS by limiting peptide aggregation and/or activating cellular stress response pathways, whereas sustained modification of Fes1 in the absence of Msr activity is detrimental to proteostasis under non‐stress conditions. Moreover, our studies with Fes1 uncovered that the two yeast Msr proteins (Mxr1 and Mxr2) are expressed in the cytoplasm, in contrast to prior studies of Mxr2 suggesting sole mitochondrial localization. We show that Mxr1 and Mxr2 co‐operate to regulate the redox state of Fes1 both in vivo and in vitro. Together, these studies provide a new perspective on how activities of the well‐studied Hsp70 family can be modulated by ROS, and further define how methionine modification is used as a means for cells to sense and respond to oxidative stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nucleotide exchange factors Fes1 and HspBP1 mimic substrate to release misfolded proteins from Hsp70.

Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco-Sjögren syndrome.

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG.

Analysis of [SWI+ ] formation and propagation events.

The budding yeast, Saccharomyces cerevisiae, harbors several prions that are transmitted as altered, heritable protein conformations. [SWI+ ] is one such prion whose determinant is Swi1, a subunit of the evolutionarily conserved chromatin-remodeling complex SWI/SNF. Despite the importance of Swi1, the molecular events that lead to [SWI+ ] prionogenesis remain poorly understood. In this study, we have constructed floccullin-promoter-based URA3 reporters for [SWI+ ] identification. Using these reporters, we show that the spontaneous formation frequency of [SWI+ ] is significantly higher than that of [PSI+ ] (prion form of Sup35). We also show that preexisting [PSI+ ] or [PIN+ ] (prion form of Rnq1), or overproduction of Swi1 prion-domain (PrD) can considerably promote Swi1 prionogenesis. Moreover, our data suggest a strain-specific effect of overproduction of Sse1 - a nucleotide exchange factor of the molecular chaperone Hsp70, and its interaction with another molecular chaperone Hsp104 on [SWI+ ] maintenance. Additionally, we show that Swi1 aggregates are initially ring/ribbon-like then become dot-like in mature [SWI+ ] cells. In the presence of [PSI+ ] or [PIN+ ], Swi1 ring/ribbon-like aggregates predominantly colocalize with the Sup35 or Rnq1 aggregates; without a preexisting prion, however, such colocalizations are rarely seen during Swi1-PrD overproduction-promoted Swi1 prionogenesis. We have thus demonstrated a complex interacting mechanism of yeast prionogenesis.

A Single Point Mutation in Mitochondrial Hsp70 Cochaperone Mge1 Gains Thermal Stability and Resistance.

Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.

Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast.

Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. InSaccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that theFES1transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally nonstressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues, or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.

The nucleotide exchange factors of Hsp70 molecular chaperones.

Molecular chaperones of the Hsp70 family form an important hub in the cellular protein folding networks in bacteria and eukaryotes, connecting translation with the downstream machineries of protein folding and degradation. The Hsp70 folding cycle is driven by two types of cochaperones: J-domain proteins stimulate ATP hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) promote replacement of Hsp70-bound ADP with ATP. Bacteria and organelles of bacterial origin have only one known NEF type for Hsp70, GrpE. In contrast, a large diversity of Hsp70 NEFs has been discovered in the eukaryotic cell. These NEFs belong to the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families. In this short review we compare the structures and molecular mechanisms of nucleotide exchange factors for Hsp70 and discuss how these cochaperones contribute to protein folding and quality control in the cell.

GrpE, Hsp110/Grp170, HspBP1/Sil1 and BAG domain proteins: nucleotide exchange factors for Hsp70 molecular chaperones.

Molecular chaperones of the Hsp70 family are key components of the cellular protein folding machinery. Substrate folding is accomplished by iterative cycles of ATP binding, hydrolysis and release. The ATPase activity of Hsp70 is regulated by two main classes of cochaperones: J-domain proteins stimulate ATPase hydrolysis by Hsp70, while nucleotide exchange factors (NEF) facilitate its conversion from the ADP-bound to the ATP-bound state, thus closing the chaperone folding cycle. Beginning with the discovery of the prototypical bacterial NEF GrpE, a large diversity of Hsp70 nucleotide exchange factors has been identified, connecting Hsp70 to a multitude of cellular processes in the eukaryotic cell. Here we review recent advances towards structure and function of nucleotide exchange factors from the Hsp110/Grp170, HspBP1/Sil1 and BAG domain protein families and discuss how these cochaperones connect protein folding with quality control and degradation pathways.

Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress.

Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target.

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

A Role for an Hsp70 Nucleotide Exchange Factor in the Regulation of Synaptic Vesicle Endocytosis

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.

Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins

Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.

Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function

Despite the growing evidence of the role of oxidative stress in disease, its molecular mechanism of action remains poorly understood. The yeast Saccharomyces cerevisiae provides a valuable model system in which to elucidate the effects of oxidative stress on mitochondria in higher eukaryotes. Dimeric yeast Mge1, the cochaperone of heat shock protein 70 (Hsp70), is essential for exchanging ATP for ADP on Hsp70 and thus for recycling of Hsp70 for mitochondrial protein import and folding. Here we show an oxidative stress-dependent decrease in Mge1 dimer formation accompanied by a concomitant decrease in Mge1-Hsp70 complex formation in vitro. The Mge1-M155L substitution mutant stabilizes both Mge1 dimer and Mge1-Hsp70 complex formation. Most important, the Mge1-M155L mutant rescues the slow-growth phenomenon associated with the wild-type Mge1 strain in the presence of H2O2 in vivo, stimulation of the ATPase activity of Hsp70, and the protein import defect during oxidative stress in vitro. Furthermore, cross-linking studies reveal that Mge1-Hsp70 complex formation in mitochondria isolated from wild-type Mge1 cells is more susceptible to reactive oxygen species compared with mitochondria from Mge1-M155L cells. This novel oxidative sensor capability of yeast Mge1 might represent an evolutionarily conserved function, given that human recombinant dimeric Mge1 is also sensitive to H2O2.

Crystallization and preliminary X-ray crystallographic analysis of the Bag2 amino-terminal domain from Mus musculus.

Bag2, an atypical member of the Bag family of Hsp70 co-chaperones, acts as both an Hsp70 nucleotide-exchange factor and an inhibitor of the Hsp70-binding E3 ubiquitin ligase CHIP (carboxyl-terminus of Hsp70-interacting protein). The amino-terminal domain of Bag2 (Bag2-NTD), which is required for inhibition of CHIP, has no sequence homologs in the PDB. Native and selenomethionyl (SeMet) forms of Bag2-NTD were crystallized by hanging-drop vapor diffusion. Native Bag2-NTD crystals diffracted to 2.27 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 75.5, b = 84.7, c = 114.1 Å. SeMet Bag2-NTD crystals diffracted to 3.10 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 53.3, c = 86.7 Å. Phases for the SeMet Bag2-NTD crystal were solved by single-wavelength anomalous diffraction. Initial phasing and model building using the 3.10 Å resolution SeMet Bag2-NTD data set suggested that Bag2-NTD forms a dimer and adopts a fold distinct from those of any domains annotated in the Pfam or SMART domain databases.

Molecular and functional characterization of the only known hemiascomycete ortholog of the carboxyl terminus of Hsc70-interacting protein CHIP in the yeast Yarrowia lipolytica

The carboxyl terminus of Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone and a U-box ubiquitin ligase that plays a crucial role in protein quality control in higher eukaryotes. The yeast Yarrowia lipolytica is the only known hemiascomycete where a CHIP ortholog is found. Here, we characterize Y. lipolytica's CHIP ortholog (Yl.Chn1p) and document its interactions with components of the protein quality control machinery. We show that Yl.Chn1p is non-essential unless Y. lipolytica is severely stressed. We sought for genetic interactions among key components of the Y. lipolytica protein quality control arsenal, including members of the Ssa-family of Hsp70 molecular chaperones, the Yl.Bag1p Hsp70 nucleotide exchange factor, the Yl.Chn1p and Yl.Ufd2p U-box ubiquitin ligases, the Yl.Doa10p and Yl.Hrd1p RING-finger ubiquitin ligases, and the Yl.Hsp104p disaggregating molecular chaperone. Remarkably, no synthetic phenotypes were observed among null alleles of the corresponding genes in most cases, suggesting that overlapping pathways efficiently act to enable Y. lipolytica cells to survive under harsh conditions. Yl.Chn1p interacts with mammalian and Saccharomyces cerevisiae members of the Hsp70 family in vitro, and these interactions are differently regulated by Hsp70 co-chaperones. We demonstrate notably that Yl.Chn1p/Ssa1p interaction is Fes1p-dependent and the formation of an Yl.Chn1p/Ssa1p/Sse1p ternary complex. Finally, we show that, similar to Sse1p, Yl.Chn1p can act as a "holdase" to prevent the aggregation of a heat-denatured protein.