Putative Protein Interactome of the Rhomboid Protease RHBDL4.

The physiological functions of the rhomboid-related protein 4 (RHBDL4) are emerging, but their molecular details remain unclear. Because increased expression of RHBDL4 has been clinically linked to poorer outcomes in cancer patients, this association urgently demands a better understanding of RHBDL4. To elucidate the molecular interactions and pathways that RHBDL4 may be involved in, we conducted proximity-dependent biotin identification (BioID) assays. Our analyses corroborated several of the expected protein interactors such as the transitional endoplasmic reticulum (ER) ATPase VCP/p97 (TERA), but they also described novel putative interactors including IRS4, PGAM5, and GORS2. Using proximity-ligation assays, we validated VCP/p97, COPB, and VRK2 as proteins that are in proximity to RHBDL4. Overall, our results support the emerging functions of RHBDL4 in ER quality control and also point toward putative RHBDL4 functions in protein membrane insertion and membrane organization and trafficking.

Sorghum bicolor SbHSP110 has an elongated shape and is able of protecting against aggregation and replacing human HSPH1/HSP110 in refolding and disaggregation assays.

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.

The J Domain of Sacsin Disrupts Intermediate Filament Assembly.

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS) is caused by mutation in the SACS gene resulting in loss of function of the protein sacsin. A key feature is the formation of abnormal bundles of neurofilaments (NF) in neurons and vimentin intermediate filaments (IF) in cultured fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons. Having studied the mechanism using NF assembled in vitro from purified NF proteins, we report that the SacsJ domain interacts with NF proteins to disassemble NFL filaments, and to inhibit their initial assembly. A cell-penetrating peptide derived from this domain, SacsJ-myc-TAT was efficient in disassembling NF bundles in cultured Sacs-/- motor neurons, restoring the NF network; however, there was some loss of vimentin IF and NF in cultured Sacs+/+ fibroblasts and motor neurons, respectively. These results suggest that sacsin through its SacsJ domain is a key regulator of NF and vimentin IF networks in cells.

BAG3P215L/KO Mice as a Model of BAG3P209L Myofibrillar Myopathy.

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

Membrane cholesterol as regulator of human rhomboid protease RHBDL4.

In the last decade, intramembrane proteases have gained increasing attention because of their many links to various diseases. Nevertheless, our understanding as to how they function or how they are regulated is still limited, especially when it comes to human homologues. In this regard, here we sought to unravel mechanisms of regulation of the protease rhomboid-like protein-4 (RHBDL4), one of five active human serine intramembrane proteases. In view of our recent finding that human RHBDL4 efficiently cleaves the amyloid precursor protein (APP), a key protein in the pathology of Alzheimer's disease, we used established reagents to modulate the cellular cholesterol content and analyzed the effects of this modulation on RHBDL4-mediated processing of endogenous APP. We discovered that lowering membrane cholesterol levels increased the levels of RHBDL4-specific endogenous APP fragments, whereas high cholesterol levels had the opposite effect. Direct binding of cholesterol to APP did not mediate these modulating effects of cholesterol. Instead, using homology modeling, we identified two potential cholesterol-binding motifs in the transmembrane helices 3 and 6 of RHBDL4. Substitution of the essential tyrosine residues of the potential cholesterol-binding motifs to alanine increased the levels of endogenous APP C-terminal fragments, reflecting enhanced RHBDL4 activity. In summary, we provide evidence that the activity of RHBDL4 is regulated by cholesterol likely through a direct binding of cholesterol to the enzyme.

Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein.

The discovery of proteases and intramembrane proteolysis 1.

Proteases carry out a wide variety of physiological functions. This review presents a brief history of protease research, starting with the original discovery of pepsin in 1836. Following the path of time, we revisit how proteases were originally classified based on their catalytic mechanism and how chemical and crystallographic studies unravelled the mechanism of serine proteases. Ongoing research on proteases addresses their biological roles, small molecule inhibitors for therapeutic uses, and protein engineering to modify their activities. The discovery of intramembrane proteases is more recent, beginning with the discovery of site-2 protease in 1997. Since then, different mechanistic classes of intramembrane proteases have been characterized, and many of these act in regulated intramembrane proteolysis in signaling pathways. Furthermore, the rhomboid intramembrane proteases were discovered by genetic and biochemical experiments in Drosophila and then in human cells. Research on the intramembrane proteases is expanding, as their biological importance is recognized.

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.

Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70.

To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.

hERG quality control and the long QT syndrome.

Long-QT syndrome type-2 (LQT2) is characterized by reduced functional expression of the human ether-à-go-go related (hERG) gene product, resulting in impaired cardiac repolarization and predisposition to fatal arrhythmia. Previous studies have implicated abnormal trafficking of misfolded hERG as the primary mechanism of LQT2, with misfolding being caused by mutations in the hERG gene (inherited) or drug treatment (acquired). More generally, environmental and metabolic stresses present a constant challenge to the folding of proteins, including hERG, and must be countered by robust protein quality control (QC) systems. Disposal of partially unfolded yet functional plasma membrane (PM) proteins by protein QC contributes to the loss-of-function phenotype in various conformational diseases including cystic fibrosis (CF) and long-QT syndrome type-2 (LQT2). The prevalent view has been that the loss of PM expression of hERG is attributed to biosynthetic block by endoplasmic reticulum (ER) QC pathways. However, there is a growing appreciation for protein QC pathways acting at post-ER cellular compartments, which may contribute to conformational disease pathogenesis. This article will provide a background on the structure and cellular trafficking of hERG as well as inherited and acquired LQT2. We will review previous work on hERG ER QC and introduce the more novel view that there is a significant peripheral QC at the PM and peripheral cellular compartments. Particular attention is drawn to the unique role of the peripheral QC system in acquired LQT2. Understanding the QC process and players may provide targets for therapeutic intervention in dealing with LQT2.

The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding.

DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.

Systems-level analyses of protein-protein interaction network dysfunctions via epichaperomics identify cancer-specific mechanisms of stress adaptation.

Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.

Interaction between the human mitochondrial import receptors Tom20 and Tom70 in vitro suggests a chaperone displacement mechanism.

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.

Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site.

Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.

OEP61 is a chaperone receptor at the plastid outer envelope.

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.

Hsp40 chaperones promote degradation of the HERG potassium channel.

Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.

Human mitochondrial import receptor Tom70 functions as a monomer.

The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer membrane 70) interacts with chaperone-preprotein complexes through two domains: one that binds Hsp70 (heat-shock protein 70)/Hsc70 (heat-shock cognate 70) and Hsp90, and a second that binds preproteins. The oligomeric state of Tom70 has been controversial, with evidence for both monomeric and homodimeric forms. In the present paper, we report that the functional state of human Tom70 appears to be a monomer with mechanistic implications for its function in mitochondrial protein import. Based on analytical ultracentrifugation, cross-linking, size-exclusion chromatography and multi-angle light scattering, we found that the soluble cytosolic fragment of human Tom70 exists in equilibrium between monomer and dimer. A point mutation introduced at the predicted dimer interface increased the percentage of monomeric Tom70. Although chaperone docking to the mutant was the same as to the wild-type, the mutant was significantly more active in preprotein targeting. Cross-linking also demonstrated that the mutant formed stronger contacts with preprotein. However, cross-linking of full-length wild-type Tom70 on the mitochondrial membrane showed little evidence of homodimers. These results indicate that the Tom70 monomers are the functional form of the receptor, whereas the homodimers appear to be a minor population, and may represent an inactive state.

Volunteer geographic information in the Global South: barriers to local implementation of mapping projects across Africa.

The world is awash in data-by 2020 it is expected that there will be approximately 40 trillion gigabytes of data in existence, with that number doubling every 2 to 3 years. However, data production is not equal in all places-the global data landscape remains heavily concentrated on English-speaking, urban, and relatively affluent locations within the Global North. This inequality can contribute to new forms of digital and data colonialism. One partial solution to these issues may come in the form of crowdsourcing and volunteer geographic information (VGI), which allow Global South populations to produce their own data. Despite initial optimism about these approaches, many challenges and research gaps remain in understanding the opportunities and barriers that organizations endemic to the Global South face in carrying out their own sustainable crowdsourcing projects. What opportunities and barriers do these endemic organizations face when trying to carry out mapping projects driven by their own goals and desires? This paper contributes answers to this question by examining a VGI project that is currently mapping public libraries across the African continent. Our findings highlight how dramatically digital divides can bias crowdsourcing results; the importance of local cultural views in influencing participation in crowdsourcing; and the continued importance of traditional, authoritative organizations for crowdsourcing. These findings offer important lessons for researchers and organizations attempting to develop their own VGI projects in the Global South.

The chaperone HSPB1 prepares protein aggregates for resolubilization by HSP70.

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.

Mechanisms of the Hsp70 chaperone system.

Molecular chaperones of the Hsp70 family have diverse functions in cells. They assist the folding of newly synthesized and stress-denatured proteins, as well as the import of proteins into organelles, and the dissociation of aggregated proteins. The well-conserved Hsp70 chaperones are ATP dependent: binding and hydrolysis of ATP regulates their interactions with unfolded polypeptide substrates, and ATPase cycling is necessary for their function. All cellular functions of Hsp70 chaperones use the same mechanism of ATP-driven polypeptide binding and release. The Hsp40 co-chaperones stimulate ATP hydrolysis by Hsp70 and the type 1 Hsp40 proteins are conserved from Escherichia coli to humans. Various nucleotide exchange factors also promote the Hsp70 ATPase cycle. Recent advances have added to our understanding of the Hsp70 mechanism at a molecular level.