Direct evidence that the glucocorticoid receptor binds to hsp90 at or near the termination of receptor translation in vitro

We have translated the rat glucocorticoid receptor in both reticulocyte lysate and in wheat germ extract. Receptor synthesized in the reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the 90-kDa heat shock protein (hsp90) and it has a normal ability to bind glucocorticoid in a high affinity manner. Although the wheat germ extract synthesizes the full length receptor, the receptor is not immunoadsorbed by 8D3 and we cannot demonstrate high affinity steroid binding. Receptor synthesized by the reticulocyte lysate can be immunoadsorbed by antibody directed against hsp90 as soon as the translation product is full length, suggesting that the receptor becomes associated with hsp90 late during translation or immediately at the termination of translation. When newly synthesized receptor is bound with steroid and incubated at 25 degrees C, it is converted to a form that binds to DNA. This study provides direct evidence that association of hsp90 with the glucocorticoid receptor is a very early event and that the newly formed heteromeric receptor-hsp90 complex is fully competent to undergo transformation.     

Primary sequence and heterologous expression of nuclear pore glycoprotein p62

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.     

Stop-transfer activity of hydrophobic sequences depends on the translation system

Signal and stop-transfer sequences are the known determinants involved in topogenesis of integral membrane proteins. To study the characteristics of stop-transfer sequences, artificial proteins have been created on the DNA level based on the cDNA of the asialoglycoprotein receptor H1. Its internal signal/anchor domain initiates translocation of the downstream sequence across the endoplasmic reticulum membrane. The ability of several hydrophobic sequences inserted into the translocating polypeptide to stop further transfer was analyzed by translation of the fusion proteins using the wheat germ extract and rabbit reticulocyte lysate systems with dog pancreas microsomes. We discovered that some of the sequences behave differently with respect to translocation across the membrane depending on the translation system. Expression of one of the fusion proteins in fibroblasts showed that the reticulocyte lysate system reflects more closely the in vivo situation than the wheat germ system. Our results suggest that in a homologous system the translating ribosomes interact with the translocation machinery and influence the termination of polypeptide transfer by hydrophobic sequences.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.     

Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate.

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.     

Cell-free synthesis, membrane integration, and glycosylation of pro-sucrase-isomaltase

Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide.     

Identification of a putative hypothalamic mRNA coding for somatostatin and of its product in cell-free translation

Poly(A)-containing RNA from rodent hypothalamic tissue has been used to direct the synthesis of polypeptides in cell-free systems derived from wheat germ extract and rabbit reticulocyte lysate in the presence of [³⁵S]-L-cysteine. Immunoprecipitation of translation products with antiserum to somatostatin followed by sodium dodecylsulfate gel electrophoresis demon-strated the existence of a 15,000 dalton polypeptide species which was displaceable by synthetic somatostatin. In addition, hybridization of fractionated hypothalamic poly(A)-RNA, blotted against DBM-paper, with a probe containing a synthetic gene for somatostatin resulted in specific hybridization of a 550 nucleotide RNA species to the probe. These results suggest that the primary translation product for hypothalamic somatostatin is a 15,000 dalton polypeptide species.     

mRNA-dependent synthesis of rat apolipoprotein E in vitro: Cotranslational processing and identification of an endoglycosidase H-sensitive glycopeptide intermediate

Total polyadenylated enriched mRNA was prepared from rat liver by guanidine-HCl extraction and oligo(dT)-cellulose chromatography. It was translated $\text{in vitro}$ in an mRNA-dependent wheat germ system and rabbit reticulocyte lysate system, using radiolabeled leucine or methionine as amino acid precursor. A product, designated preapoE, was specifically precipitated by a rabbit anti-rat apoE serum and accounted for 1.5% of the total radioactive peptides. It migrated as a single band of radioactivity on SDS gels with an apparent molecular weight similar to that of mature plasma apoE. Inclusion of dog pancreatic microsomal membranes in the translation reaction resulted in a slightly smaller product (by 500 daltons). It also converted the preapoE from an endoglycosidase H-resistant to an enzyme-sensitive species. This suggests that processing of preapoE takes place by the cotranslational removal of a signal peptide and core glycosylation of the mature protein.     

Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.

Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.     

Monocistronic translation of alfalfa mosaic virus RNAs.

The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems.     

Cloning and characterisation of a gene encoding an antiviral protein from Clerodendrum aculeatum L.

The Clerodendrum aculeatum-systemic resistence inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses [22]. We have cloned the cDNA encoding the CA-SRI from C. aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein. The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp. The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species. CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system. The CA-SRI open reading frame was expressed in an E. coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate. Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.     

Cell-free protein synthesis in lysates of Drosophila melanogaster cells.

A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

In vitro translation of infectious flacherie virus RNA in a wheat germ and a rabbit reticulocyte system

Infectious flacherie virus is an insect picornavirus isolated from the silkworm, Bombyx mori. Its RNA was found to act as an efficient mRNA in a wheat germ extract and a rabbit reticulocyte lysate translation system. In either system the sum of molecular weights of translation products far exceeded the coding capacity of the virus genome, which suggests the occurrence of proteolytic cleavage of large primary products to smaller polypeptides as reported for other picornaviruses and/or premature termination of translation. The highest molecular weight product of 200 000 (polyprotein-like product) could be translated in both systems. One of the antigenic products common to both systems had a molecular weight of 130 000, which corresponds to the sum of molecular weights of the four major viral proteins. Another product, which comigrated with viral protein 0, the largest viral structural protein in SDS-polyacrylamide gel electrophoresis, also showed antigenicity. Peptide mapping of these polypeptides showed that the two in vitro systems translated the same cistron in the viral RNA and that the smaller polypeptide was a part of the 130 000 Da product.     

Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs.     

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.     

Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.