Mosquito vitellogenin subunits originate from a common precursor

Using a cell-free translation system, we demonstrated that the two subunits of mosquito vitellogenin (VG), 200 kDa and 65 kDa, originate from a common precursor. The precursor polypeptide of 220 kDa is a translation product specific to mRNA from vitellogenic mosquitoes. In immunoprecipitation analysis, the 220-kDa polypeptide was recognized by monoclonal antibodies directed either to the large or the small VG subunit. Peptide mapping showed homology between the 220-kDa polypeptide and both subunits, thus providing further proof that the 220-kDa product of translation is the precursor for both VG subunits. In the presence of microsomal membranes, the molecular size of the VG precursor increased to 235 kDa suggesting this as a first step in co-translational modifications of VG.     

Translational options for the pir gene of plasmid R6K: multiple forms of the replication initiator protein pi.

The autogenously controlled pir gene of plasmid R6K was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. We have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mRNA. In extracts of cells containing this mutation two translational products (35 kDa and 30.2 kDa) have been detected. We propose that the 35-kDa polypeptide produced by the pir18 op mutation contains Trp substituted for Arg18 as the result of an opal readthrough. Translation, which results in the 30.2-kDa polypeptide, originates downstream from the UGA stop signal created by the mutation. Moreover, we realize now that the 30.2-kDa polypeptide is also produced in cells containing a wild-type (wt) pir gene. The shorter variant of the pi protein lacks replication initiation and inhibition functions, as well as autorepressor activity in vivo. We also show that an in-frame fusion of seven N-terminal codons of the trpE gene with a pir gene lacking the first two codons produces two polypeptides which replace the 35-kDa pi protein and are of similar molecular weight. Thus, at least three options exist in the translation of the wt pir mRNA. Start codons are most likely at codon positions 1, 6 or 7, and 36 or 38. Each of these five AUG codons is preceded by a consensus ribosome-binding site (RBS).     

Biosynthesis of nonspecific lipid transfer protein (sterol carrier protein 2) on free polyribosomes as a larger precursor in rat liver

The biosynthesis of nonspecific lipid transfer protein (nsLTP) was investigated. Total RNA of rat liver was translated in a rabbit reticulocyte lysate cell-free protein-synthesizing system with [35S]methionine as label. The immunoprecipitation of translation products with affinity-purified anti-nsLTP antibody yielded 14.5- and 60-kDa [35S]polypeptides. The molecular mass of the former polypeptide was approximately 1.5 kDa larger than that of the purified mature nsLTP (13 kDa). The site of synthesis of nsLTP was studied by in vitro translation of free and membrane-bound polyribosomal RNAs followed by immunoprecipitation. mRNA for both the 14.5- and 60-kDa polypeptides were found predominantly in the free polyribosomal fraction in both normal and clofibrate-treated rats. Clofibrate, a hypolipidemic drug that proliferates peroxisomes, did not increase the relative amount of nsLTP mRNA in rat liver. Pulse-chase experiments in rat hepatoma H-35 cells suggested that nsLTP was synthesized as a larger precursor of 14.5 kDa and converted to a mature form of 13 kDa. We have recently shown that nsLTP is highly concentrated in peroxisomes in rat hepatocytes [Tsuneoka et al. (1988) J. Biochem. 104, 560-564]. Taken together, these results suggest that nsLTP is synthesized as a larger precursor of 14.5 kDa on cytoplasmic free polyribosomes, then post-translationally transported to peroxisomes, where the precursor is presumably proteolytically processed to its mature form of 13 kDa. The relationship between the 13-kDa nsLTP and the 60-kDa polypeptide is also discussed.     

Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte

The pattern of protein synthesis in oocytes of starfish Marthasterias glacialis changes during 1-methyladenine-induced meiotic maturation. One of the newly synthesized proteins, a major 54-kDa polypeptide, was synthesized continuously after activation but was destroyed abruptly just before appearance of the polar bodies at each meiotic division. This protein thus resembles the cyclin proteins identified in cleaving sea urchin and clam embryos. RNA extracted from oocytes before and after maturation encoded virtually identical polypeptides when translated in the reticulocyte lysate. However, there was poor correspondence between the in vitro translation products and the labelling pattern of intact cells. There was no exact in vitro counterpart to the in vivo-labelled cyclin. Instead, a major polypeptide of 52 kDa was seen which appears to be a precursor of the 54-kDa form of cyclin. The 52-kDa polypeptide was identified as cyclin by hybrid arrest of translation. Cyclin mRNA is ot translated to a significant extent before oocyte activation and is present in oocytes as nonadenylated form. It becomes polyadenylated when the oocytes mature. This behavior is also seen in the case of the mRNA for the small subunit of ribonucleotide reductase, another abundant maternal mRNA whose translation is activated at maturation.     

Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system.

Assembly of glycosylphosphatidylinositol (GPtdIns)-anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C-terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns-anchoring reaction, we used a small reporter protein that becomes GPtdIns-anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site-specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns-anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nepsilon-(5-azido-2-nitrobenzoyl)-lysyl-tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns-attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass approximately 60 kDa, approximately 70 kDa, and approximately 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The approximately 70-kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.     

ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner

Eukaryotic initiation factor 2 (eIF2) plays a key role in the process of translation initiation and in its control. Here we demonstrate that highly purified mammalian eIF2 contains an additional polypeptide of apparent molecular mass of 110 kDa. This polypeptide co-purified with eIF2 through five different chromatography procedures. A cDNA clone encoding the polypeptide was isolated, and its sequence closely matched that of a protein previously termed ABC50, a member of the ATP-binding cassette (ABC) family of proteins. Antibodies to ABC50 co-immunoprecipitated eIF2 and vice versa, indicating that the two proteins interact. The presence of ABC50 had no effect upon the ability of eIF2 to bind GDP but markedly enhanced the association of methionyl-tRNA with the factor. Unlike the majority of ABC proteins, which are membrane-associated transporters, ABC50 associates with the ribosome and co-sediments in sucrose gradients with the 40 and 60 S ribosomal subunits. The association of ABC50 with ribosomal subunits was increased by ATP and decreased by ADP. ABC50 is related to GCN20 and eEF3, two yeast ABC proteins that are not membrane-associated transporters and are instead implicated in mRNA translation and/or its control. Thus, these data identify ABC50 as a third ABC protein with a likely function in mRNA translation, which associates with eIF2 and with ribosomes.     

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.     

Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

The 23-kDa polypeptide from Common Frog lens: isolation,characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.     

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.