A single gene codes for two forms of rat nucleolar protein B23 mRNA

Protein B23 (38 kDa, pI = 5.1) is an abundant RNA-associated nucleolar phosphoprotein and putative ribosome assembly factor. A full length cDNA clone (lambda JH1) encoding a major expressed form of rat protein B23, now designated B23.1, was reported recently (Chang, J. H., Dumbar, T. S., and Olson, M. O. J. (1988) J. Biol. Chem. 263, 12824-12837). In this paper the isolation from a rat brain library and sequence of a cDNA clone (lambda JH2) coding for a second form (B23.2) of protein B23 is reported. Isoforms B23.1 and B23.2 are polypeptides of 292 and 257 amino acids, respectively. The 5'-untranslated regions of the two cDNAs and the amino-terminal 255 amino acids of the proteins are identical in the two isoforms. However, the 3'-untranslated regions of the mRNAs are completely different, and the dipeptide Gly-Gly in B23.1 (residues 256 and 257) is replaced by Ala-His in B23.2 indicating that the former is not a precursor of the latter. The finding of AGGT sequences in the 3' regions of lambda JH1 suggest the presence of intron-exon boundaries at the point where the two cDNAs begin to differ. To investigate the origin of the two isoforms, two rat genomic libraries were screened with oligonucleotide probes based on sequences from the unique regions of the two cDNAs. One of the genomic clones isolated (lambda JH125) contained a 6.5-kilobase fragment encoding the 3' end of both cDNAs. lambda JH125 contains four exons designated W, X, Y, and Z in the order indicated. Exons W and X encode 36 amino acids at the carboxyl terminus of B23.2, whereas exons W, Y, and Z encode the carboxyl-terminal 71 amino acid residues of B23.1. Exons X and Z each contain distinct 3'-untranslated sequences in which are found polyadenylation signals. These data suggest that two different mRNAs are formed by alternative splicing of separate 3' segments onto a common 5' region.     

DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli

Using a cDNA probe encoding the nucleolar protein N038 of Xenopus laevis, we have isolated clones that code for the corresponding mammalian protein from cDNA libraries of mouse embryonal carcinoma and fetal liver cells. The murine cDNA-derived amino acid sequence defines a polypeptide of 292 amino acids (including the initial methionine) of a total molecular weight of 32560 and identifies a single 1.5 kb mRNA on Northern blot hybridization. This polypeptide, which is highly homologous to the Xenopus protein N038, displays an organization in three major domains: (1) an aminoterminal portion of 119 amino acids, which shows a striking homology to nucleoplasmin of Xenopus; (2) a central portion of 68 amino acids that contains two extended acidic domains, a shorter of 13 residues and a longer of 29 residues, separated by an interval enriched in positively charged amino acids; (3) a carboxyterminal portion of 105 amino acids, which is almost identical to the reported partial amino acid sequence of human and rat nucleolar protein termed B23. The sequence comparisons show that the murine protein is the mammalian counterpart to the nucleolar protein N038 of Xenopus and is compatible with the idea that both proteins N038 represent the amphibian and murine equivalents to the human and rat nucleolar phosphoprotein B23. Special sequence features and predicted conformations of this protein are discussed in relation to the specific localization and the possible functions of this major nucleolar protein.     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum

A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.     

Amino acid sequence and sites of phosphorylation in a highly acidic region of nucleolar nonhistone protein C23.

Novikoff hepatoma nucleolar nonhistone proteins, C23 and B23, contain highly acidic phosphorylated regions (Mamrack, M. D., et al. (1977) Biochem. Biophys. Res. Commun. 76, 150--157). Tryptic peptides from protein C23 containing these regions were purified by DEAE-Sephadex columns and paper electrophoresis at pH 1.8. One of these, peptide C23-Ca, was sequenced by combined automated and conventional methods. The proposed amino acid sequence is shown in eq 1. This peptide was found in three 32P-labeled forms with phosphoryl groups at positions 8 and 25, and probably 28. The highly acidic sequences adjacent to the phosphorylation sites represent a unique class of phosphorylation sites different from those in histones or substrates for cytoplasmic cAMP-dependent kinases. Ala-Ala-Pro-Ala-A5la-Pro-Ala-Ser-Glu-A10sp-Glu-Asp-Glu-Glu-A15sp-Asp-Asp-Asp-Glu-A20sp-Asp-Asp-Asp-Asp-S25er-Gln-Glu-Ser-Glu-G30lu-Glu-Asp-Glu-Glu-V35al-Met-Glu-Ile-Thr-P40ro-Ala-Lys (1).     

Mapping the functional domains of nucleolar protein B23

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.     

Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.     

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain

We have purified a novel GTP-binding protein (G protein) with a Mr of about 24,000 to homogeneity from bovine brain membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In the present studies, we have isolated and sequenced the cDNA of this G protein from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences. The cDNA of the G protein has an open reading frame encoding a protein of 220 amino acids with a calculated Mr of 24,954. This G protein is designated as the smg-25A protein (smg p25A). The amino acid sequence deduced from the smg-25A cDNA contains the consensus sequences of GTP-binding and GTPase domains. smg p25A shares about 28 and 44% amino acid homology with the ras and ypt1 proteins, respectively. In addition to this cDNA, we have isolated two other homologous cDNAs encoding G proteins of 219 and 227 amino acids with calculated Mr values of 24,766 and 25,975, respectively. These G proteins are designated as the smg-25B and smg-25C proteins (smg p25B and smg p25C), respectively. The amino acid sequences deduced from the three smg-25 cDNAs are highly homologous with one another in the overall sequences except for C-terminal 32 amino acids. Moreover, three smg p25s have a consensus C-terminal sequence, Cys-X-Cys, which is different from the known C-terminal consensus sequences of the ras and ypt1 proteins, Cys-X-X-X and Cys-Cys, respectively. These results together with the biochemical properties of smg p25A described previously indicate that three smg p25s constitute a novel G protein family.     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

The partial amino acid sequence of bovine cartilage proteoglycan, deduced from a cDNA clone, contains numerous Ser-Gly sequences arranged in homologous repeats.

We have determined the sequence of a partial cDNA clone encoding the C-terminal region of bovine cartilage aggregating proteoglycan core protein. The deduced amino acid sequence contains a cysteine-rich region which is homologous with chicken hepatic lectin. This lectin-homologous region has previously been identified in rat and chicken cartilage proteoglycan. The bovine sequence presented here is highly homologous with the rat and chicken amino acid sequences in this apparently globular region. A region containing clusters of Ser-Gly sequences is located N-terminal to the lectin homology domain. These Ser-Gly-rich segments are arranged in tandemly repeated, approx. 100-residue-long, homology domains. Each homology domain consists of an approx. 75-residue-long Ser-Gly-rich region separated by an approx. 25-residue-long segment lacking Ser-Gly dipeptides. These dipeptides are arranged in 10-residue-long segments in the 100-residue-long homology domains. The shorter homologous segments are tandemly repeated some six times in each 100-residue-long homology domain. Serine residues in these repeats are potential attachment sites for chondroitin sulphate chains.