The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG‐1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG‐1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy‐terminal peptide‐binding domain, and can be co‐immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG‐1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG‐1 inhibits Hsp/Hsc70‐mediated in vitro refolding of an unfolded protein substrate, whereas BAG‐1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG‐1 to one of its known cellular targets, Bcl‐2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG‐1 also protected certain cell lines from heat shock‐induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG‐1 may explain the diverse interactions observed between BAG‐1 and several other proteins, including Raf‐1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG‐1 on Hsp/Hsc70 chaperone activity suggest that BAG‐1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.

3Corresponding author reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp7O function and intermolecular interactions with HDJ-1.

Many biological processes are initiated by cooperative assembly of large multicomponent complexes; however, mechanisms for modulating or terminating the actions of these complexes are not well understood. For example, hormone-bound intracellular receptors (IRs) nucleate formation of transcriptional regulatory complexes whose actions cease promptly upon hormone withdrawal. Here, we show that the p23 molecular chaperone localizes in vivo to genomic response elements in a hormone-dependent manner, disrupting receptor-mediated transcriptional activation in vivo and in vitro; Hsp90 weakly displayed similar activities. Indeed, p23 and Hsp90 also disrupted the activities of some non-IR-containing transcriptional regulatory complexes. We suggest that molecular chaperones promote disassembly of transcriptional regulatory complexes, thus enabling regulatory machineries to detect and respond to signaling changes.

The properties of molecular chaperones in protein‐assisted refolding were examined in vitro using recombinant human cytosolic chaperones hsp90, hsc70, hsp70 and hdj‐1, and unfolded beta‐galactosidase as the substrate. In the presence of hsp70 (hsc70), hdj‐1 and either ATP or ADP, denatured beta‐galactosidase refolds and forms enzymatically active tetramers. Interactions between hsp90 and non‐native beta‐galactosidase neither lead to refolding nor stimulate hsp70‐ and hdj‐1‐dependent refolding. However, hsp90 in the absence of nucleotide can maintain the non‐native substrate in a ‘folding‐competent’ state which, upon addition of hsp70, hdj‐1 and nucleotide, leads to refolding. The refolding activity of hsp70 and hdj‐1 is effective across a broad range of temperatures from 22 degrees C to 41 degrees C, yet at extremely low (4 degrees C) or high (>41 degrees C) temperatures refolding activity is reversibly inhibited. These results reveal two distinct features of chaperone activity in which a non‐native substrate can be either maintained in a stable folding‐competent state or refolded directly to the native state; first, that the refolding activity itself is temperature sensitive and second, that hsp90, hsp70 (hsc70) and hdj‐1 each have distinct roles in these processes.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.

Cdc37 is required for cyclin-dependent kinase activation and is genetically linked with the activity of several other kinases, including oncogenic v-Src, casein kinase II, MPS-1 kinase, and sevenless. Strikingly, many pathways involving Cdc37 also involve the protein chaperone Hsp90. The identification of Cdc37 as the 50-kD protein in several Hsp90-kinase complexes, together with other data, led to the recent suggestion that Cdc37 is a kinase-targeting "subunit" of Hsp90. We directly examined the effect of Cdc37 on Hsp90 functions. Rather than simply acting as an accessory factor for Hsp90, Cdc37 is itself a protein chaperone with properties remarkably similar to those of Hsp90. In vitro, Cdc37 maintains denatured [3-galactosidase in an activation-competent state without reactivating it and stabilizes mature, but unstable, casein kinase II. In vivo, Cdc37 overexpression can compensate for decreased Hsp90 function, but the proteins are not interchangeable. Cdc37 can compensate for Hspg0 in maintaining the activity of v-Src kinase but does not maintain the activity of the glucocorticoid receptor. Thus, the very similar chaperone activities of the two proteins, uncovered through in vitro analysis, diverge in vivo in specific signal transduction pathways.

SUMMARY Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.