General organization of protein in HeLa 40S nuclear ribonucleoprotein particles

The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein-RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).     

Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca(2+)-binding protein abundant in the plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains in the COOH-terminal half and a putative alpha-helix domain in the NH(2)-terminal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca(2+)-binding properties. Recombinant proteins of CBP40 bound 4 mol of Ca(2+) with much higher affinity (pCa(1/2) = 6.5) than that of calmodulin. When residues 1-196 of the alpha-helix domain were deleted, the affinity for Ca(2+) decreased to pCa(1/2) = 4.6. A chimeric calmodulin was generated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca(2+) for the chimeric calmodulin was higher than that for calmodulin, suggesting that the alpha-helix domain is responsible for the high affinity of CBP40 for Ca(2+). CBP40 forms large aggregates reversibly in a Ca(2+)-dependent manner. A mutant protein with a deletion of NH(2)-terminal 32 residues, however, could not aggregate, indicating the importance of these residues for the aggregation. The aggregation occurs above micromolar levels of Ca(2+) concentration, so it may only occur when CBP40 is secreted out of the plasmodial cells.     

Metal-free and Ca2+-bound structures of a multidomain EF-hand protein,CBP40, from the lower eukaryote Physarum polycephalum

Acellular slime mold, Physarum polycephalum, has a unique wound-healing system. When cytoplasm of plasmodia is exposed to extracellular fluid, calcium binding protein 40 (CBP40) seals damaged areas, forming large aggregates Ca(2+) dependently. Part of the CBP40 is truncated at the N terminus by a proteinase in plasmodia (CBP40delta), which does not aggregate in the Ca(2+)-bound form. Here we report the crystal structures of CBP40delta in both the metal-free and the Ca(2+)-bound states. Both structures consist of three domains: coiled-coil, intervening, and EF-hand. The topology of the EF-hand domain is similar to that of calpain. The N-terminal half of CBP40Delta interacts with the C-terminal EF-hands through a large hydrophobic interface, necessary for high Ca(2+) affinity. Conformational change upon Ca(2+) binding is small; however, the structure of CBP40delta provides novel insights into the mechanism of Ca(2+)-dependent oligomerization.     

Changes in phosphorylation of nonhistone proteins during differentiation of a lower eukaryote Physarum polycephalum

During starvation-induced differentiation of a slime mold Physarum polycephalum several changes in the phosphorylation of nuclear proteins occur. The overall content of serine- and threonine-bound phosphate drops by 50% and de novo phosphorylation of a number of nonhistone proteins is drastically altered. On the contrary, no selective dephosphorylation of nuclear proteins phosphorylated under normal growth accompanies differentiation.     

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.     

Isolation and characterization of the predominant protein in nuclear ribonucleoprotein particles from rat liver

The predominant protein of the nuclear ribonucleoprotein particles of rat liver was isolated by polyacrylamide gel electrophoresis. The polypeptide represented 35% to 40% of the total mass of the protein moiety. Its molecular weight was estimated to be 38 000 and its NH2-terminal residue was found to be threonine. The amino acid composition is unique in having a high content of glycyl residues (20%) and NG-dimethylarginine (14% of total arginyl residues).     

Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles.

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.     

Identification and characterization of the packaging proteins of core 40S hnRNP particles

The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C₁ and C₂ of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A₁ and A₂ of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N^G,N^G-dimethylarginine. The major particle proteins (A₁ and A₂) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A₁, A₂ and B₁) is consistent with a conserved particle structure and function in eucaryotes.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2.

The C proteins (C1 and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S.F. Barnett, W.M. LeStourgeon, and D.L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume of 0.70 cm3/g based on the amino acid composition, the molecular weight of the complex was calculated to be 135,500. This corresponds well to 129,056, the sequence-determined molecular weight of a (C1)3C2 tetramer. Reversible chemical cross-linking with dithiobis(succinimidyl propionate) and analysis of cross-linked and cleaved complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the C proteins exist as tetramers, most or all of which are composed of (C1)3C2. The tetramer is stable in a wide range of NaCl concentrations (0.09 to 2.0 M) and is not dissociated by 0.5% sodium deoxycholate. This stability is not the result of disulfide bonds or interactions with divalent cations. The hydrodynamic properties of highly purified C-protein tetramers are the same for C-protein complexes released from intact particles with RNase or high salt. These findings support previous studies indicating that the core particle protein stoichiometry of 40S heterogeneous nuclear ribonucleoproteins is N(3A1-3A2-1B1-1B2-3C1-1C2), where N = 3 to 4, and demonstrate that the C-protein tetramer is a fundamental structural element in these RNA-packaging complexes. The presence of at least three tetramers per 40S monoparticle, together with the highly anisotropic nature of the tetramer, suggesting that one-third of the 700-nucleotide pre-mRNA moiety packaged in monoparticles is associated through a sequence-independent mechanism with the C protein.     

Identification of proliferation-sensitive human proteins amongst components of the 40 S hnRNP particles. Identity of hnRNP core proteins in the HeLa protein catalogue

The core proteins of HeLa 40 S hnRNP monoparticles have been identified in the HeLa protein catalogue. Human proteins previously identified as proliferation-sensitive [NEPHGE 21 and 17; Bravo, R. and Celis, J.E. (1982) Clin. Chem. 28, 766], as well as two proteins characterized in this study (NEPHGE 16 W and 16 W1), are shown to be components of these particles. These basic nuclear polypeptides correspond to core proteins A1, B1a, B2 and C4, respectively. The significance of these results in terms of composition and function of hnRNP particles is discussed.     

The core proteins A2 and B1 exist as (A2)3B1 tetramers in 40S nuclear ribonucleoprotein particles.

The six "core" proteins of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) package 700-nucleotide lengths of pre-mRNA into a repeating array of regular particles. We have previously shown that the C proteins exist as anisotropic tetramers of (C1)3C2 in 40S hnRNP particles and that each particle probably contains three such tetramers. We report here that proteins A2 and B1 also exist in monoparticles as (A2)3B1 tetramers and that each monoparticle contains at least three such tetramers. Proteins A2 and B1 dissociate from isolated monoparticles as a stable tetramer upon nuclease digestion. In low-salt gradients, the tetramers sediment at 6.8S, which is consistent with a mass of 145 kDa. In 200 mM salt, the concentration which dissociates these proteins from RNA, only 4.2S dimers exist in solution. Tetramers of (A2)3B1 possess the ability to package multiples of 700 nucleotides of RNA in vitro into an array of regular, 22.5-nm 43S particles. Unlike the in vitro assembly of intact 40S hnRNP, the (A2)3B1 tetramers assemble by means of a highly cooperative process. These findings indicate that the (A2)3B1 tetramers play a major role in hnRNP assembly and they further support the contention that 40S monoparticles are regular structures composed of three copies of three different tetramers, i.e., 3[(A1)3B2, (A2)3B1, (C1)3C2].     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.