Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Polyamines enhance readthrough of the UGA termination codon in a mammalian messenger RNA

The polyamines spermidine and spermine stimulate the readthrough of the UGA termination codon of rabbit beta-globin mRNA when it is translated in a rabbit reticulocyte cell-free system. The other major polyamine, putrescine, does not show this effect. The polyamine induced readthrough is specific for UGA as the UAA termination codon of alpha-globin mRNA is not read through and general translational misreading errors are not occurring in the presence of spermidine or spermine. The probable mechanism of this effect and some possible regulatory implications are discussed.     

Composition, assembly and activation of the avian progesterone receptor

When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30°C), (3) presence of ATP, and (4) presence of Mg²⁺. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl₂ and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system.     

In contrast to the glucocorticoid receptor, the thyroid hormone receptor is translated in the DNA binding state and is not associated with hsp90

We have recently reported that the glucocorticoid receptor (GR) becomes bound to the 90-kDa heat shock protein (hsp90) at or near the end of receptor translation in vitro (Dalman, F. C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., Jr., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821). In this paper we compare the hsp90 binding and DNA binding activities of the thyroid hormone receptor (TR) to those of the GR after cell-free translation of the two receptors in rabbit reticulocyte lysate. In contrast to the newly translated GR, which is bound to hsp90 and must be transformed to the DNA binding state, the TR is not bound to hsp90 and is translated in its DNA binding form without any requirement for transformation. When the GR is translated in wheat germ extract, which does not contain hsp90, it is translated in its DNA binding form in the same manner as the TR synthesized in reticulocyte lysate. These observations provide direct evidence that binding of GR to hsp90 is associated with repression of its DNA binding function. The fact that the TR does not bind to hsp90 and is translated in its DNA binding form is consistent with the different behavior of this receptor with respect to classic steroid receptors in the intact cell. We propose that binding to hsp90 may account for the fact that most of the steroid receptors are recovered in the cytosolic fraction after lysis of hormone-free cells in low salt buffer whereas the hormone-free TR is recovered in tight association with the nucleus.     

Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein

The glucocorticoid receptor is present in cytosol prepared from cell extracts of nonhormone-treated cells as a large nonactivated (i.e. non-DNA binding) 9 S heteromeric complex which contains the Mr approximately 90,000 heat shock protein, hsp90. hsp90 is expressed under physiological conditions in mammalian cells and is also present in reticulocyte lysate, as assessed by Western immunoblotting using specific anti-hsp90 antibodies. We have translated glucocorticoid receptor mRNA in reticulocyte lysates. The receptor synthesized under cell-free conditions also interacts with hsp90 both in the presence and absence of ligand, as determined by sucrose gradient centrifugation. The in vitro synthesized glucocorticoid receptor does not bind to DNA-cellulose but can be converted to a DNA binding form following labeling with dexamethasone and heat treatment. Thus, the glucocorticoid receptor is synthesized in a nonactivated form under cell-free conditions. These data indicate that the 9 S glucocorticoid receptor complex found in cytosol does not represent an artifact due to cell homogenization and supports the existence in vivo of the glucocorticoid receptor-hsp90 complex.     

Translational Activity of mRNA Coding for Cytoskeletal Brain Proteins in Newborn and Adult Mice: A Comparative Study

Translational activity of mRNA coding for cytoskeletal brain proteins was used to determine the relative abundance of the mRNA in the brains of newborn and adult mice. mRNA was translated in a cell-free system containing rabbit reticulocyte factors. The products of translation were analyzed by two-dimensional gel electrophoresis and characterized by peptide map analysis. Comparison of the products of translation from newborn and from adult brain mRNA shows a 50% decrease in actin and tubulin from newborn to the adult stage. In contrast, the 70 kd neurofilament protein and glial fibrillary acidic protein show a twofold increase in the adult stage. The heat-shock protein HSP70 increases slightly (30%) whereas the brain isozyme of creatine kinase and the heat-shock protein HSP90 are three times as high in adult subjects as in newborns.     

Globin mRNA translation on Artemia salina ribosomes with components from Friend leukemia cells.

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cytst of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCl ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin. The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the alpha and beta chains of globin. The alpha:beta chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4:1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1:4, thus these two fractions appear to provide sources enriched in either alpha or beta globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethysulfoxide-stimulated Friend leukemia cells when translated in vitro produced alpha and beta chains in a ratio of 1:1. These peptides are formed in the same ratio in the intact cells.     

Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.

In intact cells, hsp70 proteins selectively complex with mutant p53. We report here that rabbit reticulocyte lysate contains hsp70 which selectively complexes with the mutant p53 translated in vitro. Hsp70 complexes with dimers and possibly monomers of p53 in a manner that requires the terminal 28 amino acids of p53. Using murine p53Val135, which is temperature-sensitive for phenotype, we demonstrate that p53-hsp70 complexes can occur after post-translational switching from wild-type to mutant p53 phenotype. Moreover, the temperature-induced switch of full-length p53Val135 from wild-type to mutant phenotype is ATP-independent, whereas the switch from mutant to wild-type form requires ATP hydrolysis and involves hsp70. These results imply that hsp70 is involved in the regulation of p53 conformation.     

Isolation and in vitro translation of human placental lactogen messenger RNA from human term placenta.

A messenger activity for HPL was identified in normal human term placentas. The mRNA was translated in rabbit reticulocyte cell-free system. The HPL synthesized was quantified by a specific immunoprecipitation and further identified by electrophoresis on sodium dodecyl sulfate polyacrylamide gel. The HPL synthesized in the reticulocyte lysate exhibited a molecular weight between 20,000 and 22,000 daltons similar to the active hormone. The messenger RNA activity for HPL corresponded to a sedimentation coefficient of 11-12 S. Furthermore the messenger activity for HPL was preferentially associated with membrane bound polyribosomes than with free polyribosomes.     

Nonuniform size distribution of nascent peptides: The role of messenger RNA

The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.     

Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.

Inhibition of protein synthesis in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this report, immunoadsorption with monoclonal antibodies (mAbs) and Western blot analysis were used to investigate the interaction of HRI, the 90-kDa heat shock protein (hsp 90), hsp 70, and the EC1 antigen in rabbit reticulocyte lysates under protein synthesizing conditions. The data indicate that hsp 90, hsp 70, and the EC1 antigen interact with HRI in rabbit reticulocyte lysate. The EC1 antigen is a protein that has been demonstrated to be associated with several steroid hormone receptor-hsp 90 complexes and reacts with the KN 382/EC1 mAb (EC1). The association of HRI with hsp 90 and the EC1 antigen in the reticulocyte lysate was found to be dependent on the presence of hemin at a concentration of 5 microM or higher; little HRI was coadsorbed by the 8D3 anti-hsp 90 mAb or the EC1 mAb in the absence of hemin. Hsp 70 remains associated with HRI in the absence of hemin, suggesting that hsp 90 and 70 may bind to HRI at different sites. The immunological properties of the hsp 70 associated with HRI indicate that it may be the constitutively express heat shock cognate protein (hsc 73). The results suggest that the association of HRI with hsp 90 and the EC1 antigen may be in a dynamic equilibrium, in which complex formation is either facilitated or stabilized by the presence of hemin, and supports the notion that these proteins in conjunction with hsp 70 may play a role in regulating HRI activity or activation in situ.     

Assembly of progesterone receptor with heat shock proteins and receptor activation are ATP mediated events.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Cell-Free Translation of Free and Membrane-Bound Polysomes and Polyadenylated mRNA from Rabbit Brain Following Administration of d-Lysergic Acid Diethylamide In Vivo

Abstract: Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following either LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.     

A special repressor/activator system controls distribution of mRNA between translationally active and inactive mRNPs in rabbit reticulocytes

Translation of free mRNPs and polyribosomal mRNPs from rabbit reticulocytes was studied in a rabbit reticulocyte and wheat germ cell-free systems. It has been shown that translation efficiency of polyribosomal mRNPs and the mRNA isolated from the particles is nearly the same in both systems. At the same time, mRNP's translatability, which is high in the homologous cell-free system, is very low in the system from wheat germs. Translation efficiency of free mRNPs in the wheat germ system can be restored by addition of 0.5 M K CI-wash of rabbit reticulocyte ribosomes. The results testify to the existence of some special repressor repressor/activator system which controls the distribution of mRNA between free mRNPs and polyribosomes in rabbit reticulocytes.     

Characterization of A units released from the poly(A) tract of rabbit globin mRNA during protein synthesis: possible role of the released ATP in synthesizing protein

1. Rabbit globin mRNA poly(A) was translated in two cell-free synthesizing systems, rabbit reticulocyte lysate and wheat germ extract, to characterize the product released from the poly(A) tract during globin synthesis. 2. Kinetic studies indicate that the size of the cleaved nucleotide proves to be a monomer, as revealed by column chromatography on Sephadex G-100 or G-25. 3. Characterization of the monomer was accomplished by chromatography on DEAE-cellulose. Initially, 5 min post-translation, the monomer was ATP only; however, at later times ATP, ADP, AMP and adenosine were detected. 4. The two synthesizing systems differed in that globin mRNA poly(A) was translated at a faster rate in the wheat germ extract as revealed by the appearance of ATP, whereas AMP was detected sooner in the rabbit reticulocyte lysate. 5. The results indicate that the A unit released from the poly(A) tract during mRNA poly(A) translation is a monomer, and that these metabolites may play a role in controlling protein initiation via the released ATP.     

Antigenic proteins of Eimeria maxima gametocytes: cell-free translation and detection with recovered chicken serum

RNA was extracted from isolated Eimeria maxima gametocytes and translated in a rabbit reticulocyte cell-free protein synthesis system. The major cell-free translation products from E. maxima gametocyte RNA ranged from 225 to 50 kDa, distinct and different from uninfected chicken intestine cell-free translation products. Rabbit antiserum to E. maxima gametocytes as well as recovered chicken sera specifically precipitated some of the major gametocyte cell-free products. A time course of infected intestine RNA indicated that these cell-free synthesized gametocyte antigens appear at 130 to 138 hr postinfection.