Association of p21ras with cellular polypeptides

p21ras specific antiserum was used to immunoprecipitate p21ras polypeptides from human A431 cells. In addition to p21ras, this antiserum precipitated a series of polypeptides with relative molecular weights of 150,000, 120,000, 105,000, and 50,000. The precipitation of these polypeptides was prevented by preincubation of the antiserum with an excess of purified Ras protein. These polypeptides do not share an epitope with p21ras, and two of them (120 and 150 kDa) copurify with a fraction of p21ras. The co-precipitation of p21ras with these polypeptides was detected in a variety of cell types. The pattern of the immunoprecipitates was consistently different in normal and ras-transformed cells. The 120- and 150-kDa polypeptides are phosphorylated on serine and threonine in A431 cells. Serum treatment resulted in a 2-fold increase in the phosphoserine content of the 120-kDa polypeptides.     

Synthesis of Sindbis virus nonstructural polypeptides in chicken embryo fibroblasts.

The identification of eight previously undescribed polypeptides in chicken embryo cells infected with Sindbis virus is reported. Seven of these polypeptides were distinguishable from the virus structural polypeptides and their precursors by their molecular weights and tryptic peptide maps. The eighth was closely related to pE2 (Schlesinger and Schlesinger, 1973), a precursor to one of the virus particle glycoproteins. Pulse-chase experiments and the use of an inhibitor of proteolytic cleavage allowed a division of the seven nonstructural (NS) polypeptides into three stable end products (NS p89, NS p82, and NS p60) and four precursors (p230, p215, p150, and p76). The labeling kinetics after synchronous initiation of translation indicated that synthesis of the NS polypeptides started at a single site and showed that the order of the genes coding for the NS polypeptides was (5' leads to 3') NS p60, NS p89, and NS p82. Short-pulse experiments under conditions of both synchronized and nonsynchronized translation suggested that cleavage of the primary translation product of the NS genes occurred only after its synthesis was completed and that the first cleavage removed the C-terminal polypeptide. From these and other experiments, we propose a detailed scheme for the synthesis and processing of Sindbis virus NS polypeptides.     

Core Particles of Hepatitis B Virus and Ground Squirrel Hepatitis Virus I. Relationship Between Hepatitis B Core Antigen- and Ground Squirrel Hepatitis Core Antigen-Associated Polypeptides by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Peptide Mapping

The relationships among the core antigen polypeptides of hepatitis B virus (HBV) and ground squirrel hepatitis virus (GSHV) were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. The major core antigen polypeptides of liver-derived HBV (p22) and GSHV (p20.5) shared 56% of the spots in their peptide maps. Comparison of hepatitis B core antigen (HBcAg) p19 or ground squirrel hepatitis core antigen (GSHcAg) p16.5 with their respective major polypeptides indicated that these components probably resulted from cleavage of the major polypeptide of each virus. Other polypeptides smaller than the major component of each virus were often faint on polyacrylamide gels and probably resulted from the cleavage or degradation of components larger than p22 of HBcAg or p20.5 of GSHcAg, since their peptide maps contained spots unique to these high-molecular-weight components. p26 of GSHcAg and p27.5 of HBcAg shared approximately two-thirds of the spots on their peptide maps with those of their respective major core polypeptides. Furthermore, p37.5 of GSHcAg and p40 of HBcAg shared about 60% homology with their respective major polypeptides, and also shared many of the spots that were unique to p26 of GSHcAg or p27.5 of HBcAg but were not found in the peptide map of their respective core antigen polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands larger than 40,000 daltons were variably present, and peptide mapping indicated that these were aggregates of various smaller core antigen-associated polypeptides. The results suggest that p40 of HBcAg and p37.5 of GSHcAg are the largest unique polypeptides in these core particles, and that they are encoded for by the genome of each virus. That a subset of the spots unique to p40 or p37.5 was also found in p27.5 of HBcAg or p26 of GSHcAg, respectively, as compared to the major core polypeptides, also suggests that p27.5 and p26 are unique proteins encoded by the genome of each virus. It is proposed that the core antigen gene of each virus is larger than that which would encode the major polypeptide of each virus, and that the genetic organizations of the core genes of HBV and GSHV are very similar.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Immediate induction of a 45 K secreted glycoprotein by serum and growth factors in quiescent mouse 3T3 cells : Two-dimensional gel analysis

Stimulation of quiescent 3T3 cells by serum dramatically induces the synthesis of a group of secreted polypeptides with a molecular weight (MW) of 45 K (p45 A, B, C, D). The synthesis of these polypeptides increases 10-fold during the first 2 h. Cycloheximide superinduces the 45 K polypeptides and actinomycin D (actD) blocks completely their induction by serum. Peptide mapping analysis and pulse-chase experiments revealed that p45 A is a precursor of polypeptides p45 B, C, D. Tunicamycin treatment inhibits the synthesis of all four polypeptides but a new related protein appears, p-p45, the unglycosylated precursor. In the presence of tunicamycin, p-p45 is also found in the medium, demonstrating that glycosylation is not essential for the secretion. In vitro translation experiments show that the levels of p45 mRNA present in stimulated cells are severalfold higher than that of non-stimulated cells. Purified growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) induce the synthesis of p45 in quiescent cells.     

Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.     

Serological studies with low-molecular-weight polypeptides from the Moloney strain of murine leukemia virus.

Major virion low-molecular-weight polypeptides were isolated from the Moloney strain of murine leukemia virus (type C) by agarose chromatography in 6M guanidine hydrochloride and were shown to have molecular weights of 15,000 (p15), 12,000 (p12), and 10,000 (p10) by their elution volumes and by their relative mobilities in sodium dodecyl sulfate-polyacrylamide gels. Each polypeptide could be iodinated and employed in double antibody radioimmunoassay procedures. All three polypeptides demonstrated a high degree of type-specificity in serologic immunoprecipitation analysis and in corresponding competition immunoassays. The p15 was immunologically distinct from other viron polypeptides including p12 and p10; the p12 and p10 were highly related to each other but not to other virion polypeptides and were even more type-specific than the p15 in serologic tests. Competition immunoassays with p15 and p10 indicate that the Moloney strain of MuLV is only a distant relative of the Friend-Rauscher group. The combined use of the Kirsten and Moloney low-molecular-weight polypeptide immunoassays suggest that xenotropic viruses constitute yet another group(s) of murine leukemia virus with distinct type-specific antigens, further expanding an already heterogeneous group of mouse type C viruses.     

Small RNAs in Bombyx mori nuclear polyhedrosis virus polyhedra form complexes with polypeptide p14 and polyhedrin

It was shown that two small RNAs about 65 and 55 nucleotides long included in NPV B. mori polyhedra form with polypeptides p29 and p14 specific RNP-complexes with molecular weights of 50 and 31 kDa, respectively. Both complexes form high-molecular weight complex with polyhedrin. Origin and nature of p29 and p14 polypeptides are discussed.     

Structural Studies of Avian Myeloblastosis Virus: Comparison of Polypeptides in Virion and Core Component by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.     

Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10.

Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.     

The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides

The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.     

Unique composition of plastid chaperonin-60: alpha and beta polypeptide-encoding genes are highly divergent

Molecular chaperones of the chaperonin family occur in prokaryotes and in plastids and mitochondria. Prokaryotic and mitochondrial chaperonin-60 oligomers (Cpn-60) are composed of a single subunit type (p60cpn-60). In contrast, preparations of purified plastid Cpn-60 contain approximately equal quantities of two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, with slightly different electrophoretic mobilities. We have isolated cDNA clones encoding plastid p60cpn-60 alpha and p60cpn-60 beta polypeptides from Brassica napus and Arabidopsis thaliana. The unexpected degree of sequence divergence observed between p60cpn-60 alpha and p60cpn-60 beta raises questions concerning the structure of the oligomer and the functions of these polypeptides. We have also found an amino acid sequence motif within all p60cpn-60 sequences which resembles the p10cpn-10 sequences.     

Two developmentally regulated messenger RNAs differing in their coding region may exist for the myelin-associated glycoprotein

Mouse and rat brain RNA were translated in vitro in a rabbit reticulocyte lysate translation system, and myelin-associated glycoprotein-related polypeptides were immunoprecipitated. Two products of Mr = 72,000 (p72MAG) and Mr = 67,000 (p67MAG) were specifically immunoprecipitated. These polypeptides were characterized as follows: 1) exogenously added purified myelin-associated glycoprotein (MAG) competitively eliminates their immunoprecipitation; 2) peptide maps obtained from them are very similar; 3) they are both destined to become glycoproteins, as assayed by insertion into dog pancreas microsomes and their subsequent sensitivity to endoglycosidase H. Translatable mRNA for the two polypeptides have different developmental expressions. p72MAG mRNA appears at an earlier age than p67MAG mRNA and remains the dominant MAG mRNA species throughout the period of rapid myelination. As the rate of myelination decreases, p67MAG mRNA becomes the dominant MAG mRNA species. Finally, two endoglycosidase H-sensitive polypeptides were specifically immunoprecipitated from mouse brain microsomes. Therefore, there exist two MAG proteins that differ slightly in their polypeptide structure and that are developmentally regulated. The possibility that the two polypeptides are synthesized de novo from two coordinately regulated mRNAs that differ in their coding region is discussed.     

Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit.

Cdc47p is a member of the minichromosome maintenance (MCM) family of polypeptides, which have a role in the early stages of chromosomal DNA replication. Here, we show that Cdc47p assembles into stable complexes with two other members of the MCM family, Cdc46p and Mcm3p. The assembly of Cdc47p into complexes with Cdc46p does not appear to be cell cycle regulated, making it unlikely that these interactions per se are a rate-limiting step in the control of S phase. Cdc45p is also shown to interact with Cdc47p in vivo and to be a component of high-molecular-weight MCM complexes in cell lysates. Like MCM polypeptides, Cdc45p is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae; however, Cdc45p remains in the nucleus throughout the cell cycle, whereas MCMs are nuclear only during G1. We characterize two mutations in CDC47 and CDC46 which arrest cells with unduplicated DNA as a result of single base substitutions. The corresponding amino acid substitutions in Cdc46p and Cdc47p severely reduce the ability of these polypeptides to assemble in a complex with each other in vivo and in vitro. This argues that assembly of Cdc47p into complexes with other MCM polypeptides is important for its role in the initiation of chromosomal DNA replication.     

Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos.

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.     

Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies.

The bovine leukemia virus (BLV) was purified from a chronically infected fetal lamb kidney cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this virus revealed the presence of eight distinguishable viral components with molecular weights ranging from 80,000 to 11,000. The major component is a non-glycosylated protein having a molecular weight of 24,000 (p24). At least three heavier polypeptides were found, one of them representing a glycoprotein (gp 60). In addition, four minor polypeptides with respective molecular weights of 19,000, 16,000, 13,000, and 11,000 were identified. In a complement fixation assay using naturally occurring antibodies of a leukemic cow, four polypeptides, which included gp 60, p35, p24, and p16, were found to be reactive.     

Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line.

The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.