Depletion of hsp90beta induces multiple defects in B cell receptor signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples

Members of the heat shock protein-90 (Hsp90) family are key regulators of biological processes through dynamic interaction with a multitude of protein partners. However, the transient nature of these interactions hinders the identification of Hsp90 interactors. Here we show that chemical cross-linking with ethylene glycolbis (succinimidylsuccinate), but not shorter cross-linkers, generated an abundant 240-kDa heteroconjugate of the molecular chaperone Hsp90 in different cell types. The combined use of pharmacological and genetic approaches allowed the characterization of the subunit composition and subcellular compartmentalization of the multimeric protein complex, termed p240. The in situ formation of p240 did not require the N-terminal domain or the ATPase activity of Hsp90. Utilizing subcellular fractionation techniques and a cell-impermeant cross-linker, subpopulations of p240 were found to be present in both the plasma membrane and the mitochondria. The Hsp90-interacting proteins, including Hsp70, p60Hop and the scaffolding protein filamin A, had no role in governing the formation of p240. Therefore, chemical cross-linking combined with proteomic methods has the potential to unravel the protein components of this p240 complex and, more importantly, may provide an approach to expand the range of tools available to the study of the Hsp90 interactome.     

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

The molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone/ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non-stress condition due to Hsp90-independent CHIP-mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP-mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non-stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.     

A client-binding site of Cdc37

The molecular chaperone Hsp90 is distinct from Hsp70 and chaperonin in that client proteins are apparently restricted to a subset of proteins categorized as cellular signaling molecules. Among these, many specific protein kinases require the assistance of Hsp90 and its co-chaperone Cdc37/p50 for their biogenesis. A series of Cdc37 deletion mutants revealed that all mutants capable of binding Raf-1 possess amino acid residues between 181 and 200. The 20-residue region is sufficient and, in particular, a five-residue segment (residue 191-195) is essential for binding to Raf-1. These five residues are present in one alpha helix (residues 184-199) in the middle of Cdc37, which is unexpectedly nested within the Hsp90-interacting domain of Cdc37, which was recently determined by crystallography, but does not seem to contribute to direct contact with Hsp90. Furthermore, an N-terminally truncated mutant of Cdc37 composed of residues 181-378 was shown to bind the N-terminal portion of Raf-1 (subdomains I-IV). This mutant can bind not only other Hsp90 client protein kinases, Akt1, Aurora B and Cdk4, but also Cdc2 and Cdk2, which to date have not been shown to physically interact with Cdc37. These results suggest that a region of Cdc37 other than the client-binding site may be responsible for discriminating client protein kinases from others.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

Mammalian CHORD-containing protein 1 is a novel heat shock protein 90-interacting protein

With two tandem repeated cysteine- and histidine-rich domains (designated as CHORD), CHORD-containing proteins (CHPs) are a novel family of highly conserved proteins that play important roles in plant disease resistance and animal development. Through interacting with suppressor of the G2 allele of Skp1 (SGT1) and Hsp90, plant CHORD-containing protein RAR1 (required for Mla resistance 1) plays a critical role in disease resistance mediated by multiple R genes. Yet, the physiological function of vertebrate CHORD-containing protein-1 (Chp-1) has been poorly investigated. In this study, we provide the first biochemical evidence demonstrating that mammalian Chp-1 is a novel Hsp90-interacting protein. Mammalian Chp-1 contains two CHORD domains (I and II) and one CS domain (a domain shared by CHORD-containing proteins and SGT1). With sequence and structural similarity to Hsp90 co-chaperones p23 and SGT1, Chp-1 binds to the ATPase domain of Hsp90, but the biochemical property of the interaction is unique. The Chp-1-Hsp90 interaction is independent of ATP and ATPase-coupled conformational change of Hsp90, a feature that distinguishes Chp-1 from p23. Furthermore, it appears that multiple domains of Chp-1 are required for stable Chp-1-Hsp90 interaction. Unlike SGT1 whose CS domain is sufficient for Hsp90 binding, the CS domain of Chp-1 is essential but not sufficient for Hsp90 binding. While the CHORD-I domain of Chp-1 is dispensable for Hsp90 binding, the CHORD-II domain and the linker region are essential. Interestingly, the CHORD-I domain of plant RAR1 protein is solely responsible for Hsp90 binding. The unique Chp-1-Hsp90 interaction may be indicative of a distinct biological activity of Chp-1 and functional diversification of CHORD-containing proteins during evolution.     

Regulation of the cytoplasmic quality control protein degradation pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.     

Hsp90's secrets unfold: new insights from structural and functional studies.

Hsp90 is a molecular chaperone associated with the folding of signal-transducing proteins, such as steroid hormone receptors and protein kinases. Results from recent studies have shed light on the structure of Hsp90 and have demonstrated that it can bind to and hydrolyse ATP. Hsp90 forms several discrete subcomplexes, each containing distinct groups of co-chaperones that function in folding pathways. Although Hsp90 is not generally involved in the folding of nascent polypeptide chains, there is a growing list of proteins whose activity depends on its function, including heat-shock factor. This review addresses recent developments in our understanding of the structure and function of Hsp90.     

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

Hsp90: a chaperone for protein folding and gene regulation.

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.     

Hsp90 inhibitors as novel cancer chemotherapeutic agents

Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for the stability and function of multiple mutated, chimeric and over-expressed signaling proteins that promote the growth and/or survival of cancer cells. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, ErbB2 and hypoxia-inducible factor 1α (HIF-1α). Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising antitumor activity in preclinical model systems. One Hsp90 inhibitor, 17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial. Because of the chemoprotective activity of several proteins that are Hsp90 clients, the combination of an Hsp90 inhibitor with a standard chemotherapeutic agent could dramatically increase the in vivo efficacy of the therapeutic agent.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS.Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.