Isolation and characterization of twenty-three ribosomal proteins from large subunits of yeast.

The proteins of large ribosomal subunits from Saccharomyces cerevisiae were separated into 25 fractions by chromatography on columns of carboxymethylcellulose (CMC). Twenty-three proteins were then purified from the 12 CMC fractions by filtration through Sephadex G-75, Sephadex G-100, and Sephacryl S-200, and/or by phosphocellulose column chromatography. The isolated proteins are YP 1, YP 2, YP 9, YP 11, YP 13', YP 16, YP 18, YP 26, YP 39, YP 41, YP 42, YP 42', YP 44, YP 45, YP 47', YP 52a, YP 53, YP 55, YP 59, YP 62, YP 68, YP A1, and YP A2. The molecular weight and amino acid composition of these proteins are presented.     

Overexpression in Escherichia coli, purification, and characterization of recombinant 60S ribosomal acidic proteins from Saccharomyces cerevisiae

The 60S ribosomal subunits from Saccharomyces cerevisiae contain a set of four acidic proteins named YP1alpha, YP1beta, YP2alpha, and YP2beta. The genes for each were PCR amplified from a yeast cDNA library, sequenced, and expressed in Escherichia coli cells using two expression systems. The first system, pLM1, was used for YP1beta, YP2alpha, and YP2beta. The second one, pT7-7, was used for YP1alpha. Expression in both cases was under the control of a strong inducible T7 promoter. The amount of induced recombinant proteins in the host cells was around 10 to 20% of the total soluble bacterial proteins. A new protocol for purification of all four recombinant proteins was established. The preliminary steps of purification were done by ammonium sulfate precipitation (YP1alpha, YP1beta) or NH4Cl/ethanol extraction (YP2alpha, YP2beta). The recombinant proteins were then purified to apparent homogeneity by only two steps of classical chromatographies, ion exchange (DEAE-cellulose) and gel filtration (Sephacryl S-200). Isoelectrofocusing analysis of YP2alpha and YP2beta showed the pIs of the recombinant proteins are the same as that of the native yeast ribosomal P2 proteins. The pI of YP1alpha is changed due to the addition of five amino acids attached to the N-terminus of recombinant polypeptide from the expression vector. YP1beta was obtained as a truncated form of polypeptide, similar to its ribosomal counterpart, YP1beta'. This was proved by isoelectrofocusing gel analysis.     

Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis.

The genes encoding the four acidic ribosomal phosphoproteins have been inactivated in Saccharomyces cerevisae by recombination with truncated genes carrying different genetic markers. By crossing single haploid disruptants, strains harboring two simultaneously inactivated acidic protein genes were constructed. None of the six possible double disruptions was lethal, but the simultaneous inactivation of either YP1 alpha and YP1 beta(L44') or YP2 alpha(L44) and YP2 beta(L45) caused an important decrease in the cell growth rate. Ribosomes isolated from these slow-growing strains did not contain acidic proteins, not even the two polypeptides whose genes were still intact, although these proteins were present in the cell extracts and they seem to be able to form high-molecular weight protein complexes. Transformation of a slow-growing double transformant with a plasmid containing one of the disrupted genes restored the presence of the acidic proteins in the ribosomes and normal growth rates. The particles of the slow-growing strains were active in an in vitro amino acid polymerizing system, although their activity could be stimulated by the exogenous addition of the missing proteins. These results indicate that in the absence of either YP1 alpha and YP1 beta(L44') or YP2 alpha (L44) and YP2 beta(L45), the remaining acidic proteins are unable to interact with the ribosome in a stable manner, but that a strong interaction of these ribosomal components with the particle is not an absolute requirement for in vivo and in vitro protein synthesis.     

Proteins, associated with 50S-ribosomal subunits of Escherichia coli MRE600]

The proteins associated with the ribosomal subunits having the molecular masses from 158 to 47 kDa were isolated from hyaloplasmic, nucleoid and membrane fractions of Escherichia coli MRE600 cells. The proteins are eliminated from 50S subunits of ribosomes by thrice washing with the 1 M ammonium chloride buffer. 50S subunit proteins were found to be immunologically related to the inner membrane proteins. The native 50S subunits of ribosomes possess the expressed ATP-ase activity, while the washed off subunits lose it completely.     

Relative rates of turnover of subunits of mitochondrial proteins.

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 244, 3303--3315] for the measurement of protein turnover was used to estimate the turnover rates of protein subunits from rat liver submitochondrial fractions resolved by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. NaH14CO3 and [5-3H]arginine were used as first and second precursors respectively. 2. Marked heterogeneity of protein subunit turnover rates is seen for protein subunits from water-soluble, salt-soluble and Tween 20-soluble mitochondrial proteins. 3. Much lower heterogeneity is seen in the turnover of protein subunits in Triton X-100-soluble material not binding to DEAE-cellulose at low ionic strength. The relative rates of turnover of proteins in this fraction are lower than for proteins in any other submitochondrial fraction. This fraction contains the integral membrane proteins. 4. Incorporation of [3H]arginine into subunits of the cytochrome oxidase complex is greatest for subunits with molecular weights in excess of 20000. 5. No correlation is seen between protein subunit size and the rate of turnover of the protein subunits in any of the submitochondrial fractions.     

Characterization and quantification of low molecular weight glutenins in durum wheats

Durum wheat proteins have been considered as a model because of the very clear-cut relationship previously evidenced between the electrophoretic type '42' or '45' of the components that are coded by the Gli-B1 chromosome locus and the intrinsic quality (gluten viscoelasticity) of cultivars. The proteins from 4 cultivars were subjected to sequential extraction and separated into five groups, respectively, in: NaCl, EtOH (gliadins-I), EtOH + mercaptoethanol (ME) (gliadins-II), AcOH + ME (glutenins-I) and SDS + ME (glutenins-II) and characterized using polyacrylamide gel electrophoresis (PAGE), SDS-PAGE and 2-dimensional (NEPHGE X SDS - PAGE) electrophoretic systems. EtOH-soluble fractions were also separated by ion-exchange chromatography, each fraction being characterized in PAGE and SDS-PAGE and its composition in major bands determined by densitometry. From the ratio of each chromatographic fraction and of each solubility group, an estimation of the major bands or electrophoretic zones was also made in respect to the whole proteins. In 'type 45' cultivars, it was shown that only 67% of the EtOH-soluble fraction (although considered as classical gliadins) had a monomeric character, giving rise to discrete bands in PAGE systems. The remainder (33%) were aggregated fractions, essentially those referred to as low molecular weight glutenins (LMWG), that migrate, upon reduction only, in SDS-PAGE systems. LMWG make up 27% of total proteins and are revealed as a strong triplet in the 44,500-51,500 MW region, in gliadin-I and especially in gliadin-II groups. In type '42' cultivars, the LMWG ratio is reduced about by half (18% of EtOH soluble fraction, 14% of total proteins). This difference, coupled with their aggregative behavior, leads to their consideration as the major functional markers of gluten quality, gliadins 42/45 being genetic markers only. Without excluding possible physicochemical differences between different LMWG allelic types, it is hypothesized that quantitative differences could explain by themselves the quality differences between the two durum wheat genetic types. Concerning the other aggregative fractions, like high molecular weight glutenin (HMWG) subunits in glutenin-I and II groups, they do not show (unlike bread wheats) quantitative or qualitative differences large enough to play a major role in explaining genetic differences in durum wheat gluten characteristics. It is recommended, especially for physicochemical studies of wheat quality, to rely on a protein classification based on monomeric or aggregative characteristics, instead of Osborne's scheme based only on fractionation by solubility.(ABSTRACT TRUNCATED AT 400 WORDS)     

mRNA-binding site of ribosomes at different stages of translation. II. Affinity modification of Escherichia coli ribosomes bya benzylidene derivative of AUGU6 in pre- and post-translation complexes

Affinity labeling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methyl-amino]benzylidene derivative of AUGU6 (AUGU6-[14C]CHRCl) was studied within the pretranslocational complex ribosome.AUGU6[14C]CHRCl.tRNA(fMet)(P-site).fMetPhe-tR NA(Phe)(A-site) and posttranslocational complex ribosome.AUGU6[14C]CHRCl.fMetPhe-tRNA(Phe)(P-site). Both 30S and 50S subunits were labeled within these complexes, but the extent of 30S subunit modification was 6-8-fold higher than those for 50S subunit. Ribosomal proteins of both subunits were found to be labeled preferentially. Proteins S1, S5, S11, L1 were identified to be crosslinked with AUGU6[14C]CHRCl within the pretranslocational complex and S7--within the posttranslocational complex from the data of two-dimensional electrophoresis in the polyacrylamide gel.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.     

Subcellular Distribution of Cadmium and Nickel in Chronically Exposed Wild Fish: Inferences Regarding Metal Detoxification Strategies and Implications for Setting Water Quality Guidelines for Dissolved Metals

The objective of this study was to investigate metal detoxification in chronically exposed juvenile yellow perch (YP: Perca flavescens) and to field test the commonly assumed threshold toxicity model. Fish were collected from lakes located along a cadmium (Cd) and nickel (Ni) concentration gradient. Ambient dissolved metal concentrations were measured to evaluate exposure and total hepatic metal concentrations were determined as a measure of metal bioaccumulation. Hepatic metal partitioning among potentially metal-sensitive fractions (heat-denatured proteins, organelles) and detoxified metal fractions (metallothionein) was determined after differential centrifugation of YP liver homogenates. Major proportions of hepatic Cd were found in the heat-stable cytosolic peptides and proteins fraction (HSP; including metallothioneins), whereas Ni was mainly found in the potentially metal-sensitive heat-denaturable proteins fraction (HDP). For these chronically exposed fish there was no threshold exposure concentration below which binding of Cd or Ni to the heat-denaturable protein fraction or the organelle fraction did not occur. Metal detoxification was clearly incomplete and P. flavescens was subject to some metal-related stress, as evidenced notably by endocrine perturbations. Similar subcellular partitioning results were obtained when juvenile yellow perch were transferred from a reference lake to a Cd-contaminated lake and Cd accumulation was followed over time; there was no accumulation threshold below which Cd binding to the putative metal-sensitive fractions (HDP and organelles) did not occur. The presence of Cd and Ni in these fractions, even for low exposure concentrations and low hepatic accumulation, contradicts the threshold toxicity model that underpins metal toxicology theory and that is implicitly used in setting water quality guidelines for metals. Chronically exposed YP appear to have settled for a tradeoff between the cost of turning on their detoxification apparatus at full capacity, to completely suppress metal binding to metal-sensitive sites, and the alternative cost of allowing some binding of inappropriate metals to metal-sensitive sites.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

The polypeptide structure of Vitellogenin and Vitellin from the cockroach,Leucophaea maderae

Vitellogenin (Vg) synthesized by the fat body of Leucophaea maderaeis made up of four polypeptides with molecular weights of 160,000, 105,000, 98,000, and 57,000. Other polypeptides previously reported as part of Vg are associated with other proteins. Vitellin (Vt), the yolk protein (YP) isolated from mature oocytes and from newly formed oothecae, is a protein with a sedimentation coefficient of 28s and consists of three polypeptides with molecular weights of 105,000, 85,000, and 57,000. During vitellogenesis, the YP of developing oocytes contains both Vt and a 14s component. The 14s component is made up of four polypeptides with molecular weights of 105,000, 90,000, 85,000, and 57,000. The data suggest that 14s may not be a discrete protein but rather a form in transition between Vg and Vt in which the 98,000 dalton polypeptide is converted to the 85,000 dalton polypeptide of Vt through a 90,000 dalton intermediate. The 160,000 dalton peptide of Vg does not appear to be a part of Vt. Under alkaline conditions, both the 14s component and Vt are reduced to a polypeptide with a lower sedimentation rate in sucrose gradients. When acid conditions are restored, a protein resembling 14s is obtained. This suggests that the YP is a loosely held aggregate of similar or identical proteins with a molecular weight of about 250,000.     

Homology among 3S and 7S Globulins from Cereals and Pea

The globulins from wheat caryopses were found to consist primarily of protein sedimenting at approximately 3S and 7S. These proteins displayed a molecular weight distribution similar to that of the purified vicilin-like fractions from oat and pea, with variations occurring in the isoelectric points and relative quantities of their major subunits. concanavalin A Sepharose chromatography suggested that the major polypeptides of the wheat (3S + 7S) fraction are glycosylated. Western blot analysis using antioat (3S + 7S) globulin immunoglobulin G revealed the vicilins from pea and the globulin fractions of oat, wheat, barley, rye, corn, and rice to contain immunologically homologous polypeptides. Major groups of polypeptides were shared by all the cereals and pea, including subunits of approximately 75, 50, 40 kilodaltons and 20 to 25 kilodaltons. These results indicate that legume-like 3S and 7S globulins have been conserved and are being expressed in cereals.     

Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.     

Two-dimensional gel electrophoresis of ribosomal proteins from streptomycin-sensitive and streptomycin-resistant mutants of Chlamydomonas reinhardi.

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one-dimensional and two-dimensional gel electrophoresis. One mutant was streptomycin-sensitive the other two were streptomycin-resistant, one with a Mendelian the other with a non-Mendelian pattern of inheritance. In the 30-S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50-S subunits 34 proteins. The 40-S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60-S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10 000 to 35 000. However, the 60-S subunits contain in addition a protein of molecular weight 50 000 and the 30-S subunits show 6-7 bands of molecular weights from 50 000 to 83 000. The proteins of the cytoplasmic 80-S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70-S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.     

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.