Only a small portion of the cytoplasmic progesterone receptor is associated with Hsp90 in vivo.

In cell extracts all of the nonliganded steroid receptor molecules are found as an oligomeric complex with Hsp90 and other proteins. In previous studies we have shown that Wild-type Hsp90 and progesterone receptor (PR) are located in different cell compartments (Tuohimaa et al. [1993] Proc. Natl. Acad. Sci. USA 90:5848-5852). In the present work we studied whether PR and Hsp90 can efficiently associate provided they are present in the same cell compartment. The association of Hsp90 with PR in vivo was studied by nuclear cotranslocation and immunohistochemistry with an antibody (alphaD) which can distinguish between the oligomeric and dissociated form. Upon expression of a cytoplasmic mutant of PR with Wild-type (cytoplasmic) Hsp90 and Wild-type (nuclear) PR with NLS-Hsp90 (a Hsp90 with a nuclear localization signal), we noted that the epitope of alphaD in PR was exposed in both cases. Also, in vivo crosslinking and treatment of cells with substances which stabilize the oligomeric complex in vitro were inefficient in demonstrating or inducing a similar oligomeric receptor form detectable in vitro in cell homogenates. However, when the cytoplasmic PR mutant (DeltaPR) was coexpressed with a nuclear form of Hsp90 (NLS-Hsp90), a portion of PR was cotranslocated into the nucleus. This would indicate that steroid receptors are indeed associated with Hsp90 in intact cells, but the Hsp90-associated receptor pool represents only a small portion of the receptors. This suggests that the majority of oligomeric complexes seen in cell extracts are formed during cell fractionation.     

Progesterone receptor and hsp90 are not complexed in intact nuclei

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.     

Evidence for the existence of an oligomeric, non-DNA-binding complex of the progesterone receptor in the cytoplasm

Steroid receptors are found as a hetero-oligomeric complex in cell extracts. Due to the dynamic interaction between receptor-associated proteins and receptors, it is difficult to study the oligomeric complex in living cells. Here this was attempted in cells in which the interaction was stabilized by introducing molybdate into the cells or by incubating the cells at low temperature. The complex was studied with an antibody (aD) recognizing only the dissociated form of the chicken progesterone receptor (PR) and with antibodies (PR22, PR6). Recognizing also oligomeric forms of the receptor. When wild-type chicken PR was transfected, all antibodies showed nuclear staining. Molybdate or cold treatment of cells resulted in cytoplasmic accumulation of the PR as detected with PR22/PR6. aD, however, stained predominantly the nuclear PR in treated cells. These findings suggest that when the oligomeric complex of the PR is stabilized in intact cells in vivo and then crosslinked with paraformaldehyde, a portion of the cytoplasmic receptor is seen as an oligomeric complex, whereas, in the nucleus, most, if not all receptor molecules are in dissociated form.     

Progesterone receptor does not form oligomeric (8S), non-DNA-binding complex in intact cell nuclei

We raised a polyclonal antibody, αD, against a synthetic peptide (amino acids 522–535) of chichen progesterone receptor (PR). The Sequence is located between the DNA-binding domain and the hormone-binding domain in the refion within the sequences required for stability of the oligomeric form of PR. In the immunoblot, αD reacted with both A and B forms of PR. in the sucrose gradient and dot-blot the antibody did bot recognize the so-called 8S form of PR, which is an oligomeric complex of PR and other proteins, When the oligomeric complex was dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 45 form. To study whether a similar Complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, Freeze-drying-vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear Staining was Detected independent of the fixation technique. when receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (85). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an incerased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These Results Suggest that the oligomeric complex (85), Present in tissue extracts, does not exist in intact cell nuclei. They also Call into question the propesed role of hsp90 in regulating progesterone receptor function. © Wiley-Liss, Inc.     

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.     

In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation

The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.     

CyP40, but not Hsp70, in rabbit reticulocyte lysate causes the aryl hydrocarbon receptor-DNA complex formation

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner aryl hydrocarbon receptor nuclear translocator (Arnt). The AhR-Arnt heterodimer binds to the dioxin response element (DRE) to regulate target gene expression. Using baculovirus expressed human AhR and Arnt, we showed that the formation of the ligand-dependent AhR-Arnt-DRE complex requires protein factors in vitro. Recently, we provided evidence that p23, an Hsp90-associated protein, is involved in the complex formation. The aim of this study was to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR-Arnt-DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CyP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex.     

Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.     

Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor

Receptor-associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N- and C-termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N-terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C-terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46-mediated downregulation of glucocorticoid receptor activity.     

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.     

Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA.

The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera. To obtain these monospecific antisera, portions of cloned cDNAs encoding the individual polymerase proteins (PB1, PB2, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion proteins were injected into rabbits. Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB1 and PB2 are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells. Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus. Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins. In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins. To determine whether a polymerase protein is transported into the nucleus individually, PB1 was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector. PB1 alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus. This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB1 protein and that the PB1 protein may contain an intrinsic signal(s) for nuclear transport.     

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.     

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins

Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins. All members of this family investigated so far are oligomeric complexes. For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers. This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins. To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e. the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26DeltaN). Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins. We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy. The unfolding of the dissociated Hsp26 dimer or Hsp26DeltaN, which is a dimer, requires a much higher energy. Because Hsp26DeltaN was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes. Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes. Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.     

A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins.

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-Hsp90-FKBP52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport.