A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1

Cyanobacteria are prokaryotes that carry out plant-type photosynthesis and contain several eukaryotic-type RNA-binding proteins. Using a single-stranded DNA column, a 33-kDa protein was isolated and characterized from Synechococcus sp. PCC6301. This protein of 293 amino acids is similar in overall structure to the ribosomal protein S1 found in the same species, and contains three repeated units that are highly similar to the S1 motif originally found in the ribosomal protein S1 of Escherichia coli. However, the 33-kDa protein was found not to be associated with ribosomes and its nucleic acid binding specificity is distinct from that of the ribosomal protein S1. As this protein has high affinity for both single- and double-stranded DNA, as well as for poly(G) and poly(A), we tentatively named it nucleic acid-binding protein 1 (Nbp1). Received: 8 October 1999 / Accepted: 24 January 2000     

Z-DNA-binding proteins. Identification critically depends on the proper choice of ligands

We have previously isolated from bull testis three proteins of molecular mass 31, 33, and 58 kDa that we have tentatively characterized as high affinity Z-DNA-binding proteins. This inference was based on their preferential binding to brominated poly(dG-dC).poly(dG-dC) in Z-form as opposed to the unbrominated polynucleotide in B-form (Gut, S. H., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1987) Nucleic Acids Res. 15, 9691-9705). By partial amino acid sequencing we have provisionally identified the 31- and 33-kDa proteins as members of the high mobility group 2 and 1 protein families, respectively, whereas the 58-kDa protein has so far remained unidentified (Christen, Th., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1990) FEBS Lett. 267, 139-141). In the present study, we have critically reassessed the binding specificity of these three proteins by using more natural Z- and B-DNA ligands. As such we chose supercoiled and relaxed DNA minicircles containing a d(CG)7 insert in the Z- and B-conformation, respectively. Filter binding tests and gel retardation assays performed with these ligands showed that the three testis proteins either do not discriminate between Z- and B-DNA (31- and 33-kDa proteins) or even have a preference for B-DNA (58-kDa protein). Therefore, we question the validity of using brominated poly(dG-dC).poly(dG-dC) as an indicator of Z-DNA binding.     

Isolation of a manganese-containing protein complex from photosystem II preparations of spinach

Purified 125I-labeled 33-kDa protein binds to calcium-washed photosystem II preparations at high-affinity and low-affinity binding sites. Filling 70% of the high-affinity site with 33-kDa protein induces 63% of the maximum achievable reconstitution of O2-evolving activity. When N-succinimidyl [(4-azidophenyl)dithio]propionate modified 33-kDa protein was reconstituted into Ca(II)-washed membranes under conditions that primarily filled the high-affinity site and then cross-linked to adjacent proteins by illumination of the photoaffinity label, a cross-linked protein complex was formed that could be solubilized from the membranes with sodium dodecyl sulfate. The protein complex consisted of 22-, 24-, 26-, 28-, 29-, and 31-kDa proteins cross-linked to the 33-kDa protein and contained about 3-4 mol of Mn/mol of protein.     

Changes in protein expression in p53 deleted spontaneous thymic lymphomas

By the use of high-resolution two-dimensional gel electrophoresis and computerized image analysis we investigated and compared the expression of cellular proteins from p53 positive (+/+) mouse thymocytes, p53-/- thymocytes before neoplastic transformation, and from cell lines derived from two spontaneous p53-/- thymic lymphomas, SM5 and SM7. A total of around 1500 proteins were detected on individual gels. Only changes in protein expression by a factor of 2 or more were considered. In the thymic lymphoma cells 3-5% of the proteins were found to be differentially regulated when compared with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected proteins increased their expression levels by more than 10 times from the p53+/+ to the p53-/- thymocytes and these high expression levels were also found in thymic lymphomas. The two proteins were identified by mass spectrometry as acidic ribosomal phosphoprotein P0 and a 33-kDa protein with a primary structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA.     

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.     

Characterization of an insect ferritin subunit synthesized in a cell-free system

Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28-30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28-30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.     

Identification of a membrane protein responsible for ribosome binding in rough microsomal membranes

A membrane protein fraction was obtained from rat liver rough microsomes by affinity chromatography on a concanavalin A-Sepharose column and then a chelating-Sepharose column. This protein fraction comprised about 2% of the total membrane proteins of rough microsomes and the ribosome-binding activity of ribosome-stripped rough microsomes was predominantly found in this protein fraction, as determined with a liposome assay system. To identify the essential components responsible for the ribosome binding, two approaches were employed. Trypsin treatment of liposomes reconstituted with this protein fraction resulted in the loss of the ribosome-binding activity in parallel with the loss of a dominant band, estimated Mr 34,000, in SDS-polyacrylamide gels. Next, the direct interaction between the binding sites on the membrane of reconstituted liposomes and 60S ribosomal subunits was investigated by photocrosslinking using sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (SAND). The photocrosslinked complex was formed between 60S ribosomal subunits pretreated with SAND and binding-site proteins on the membrane of the liposomes. Then, after the liposomes were solubilized, the complex was isolated by sucrose gradient centrifugation of the binding mixture. The crosslinked proteins were released from 60S ribosomal subunits by cleavage of of crosslinks with beta-ME and analyzed by SDS-polyacrylamide gel electrophoresis and 125I-autoradiography. The 34-kDa protein (p34) was the predominant component that crosslinked to the 60S ribosomal subunits and was found in proportion to the amount of 60S ribosomal subunits added to the system. The p34 was distinguishable by immunoblot analysis from urate oxidase, which is the 34-kDa protein of peroxisomal cores contaminating rough microsomes. These results suggest that the present p34 is a likely candidate molecule for the ribosome-binding activity of rough microsomes.     

Studies on proteins of animal ribosomes XXIV. Localisation of proteins in ribosomal subunits of rat liver studied by chemical substitution with p-nitrophenyl acetate and methyl acetimidate

The reaction of [³H]p-nitrophenyl acetate (NPA) or [¹⁴C]methyl acetimidate (MAI) with amino groups of ribosomal proteins from the rate has been studied.A comparison has been made between the reactivity of the proteins in situ in the ribosomal subunit with that of isolated protein mixtures.In the small subunit reactivity compared with the protein mixture was only 10–65% in the case of NPA but 45 to more than 100% in the case of MAI.In the large subunit reactivity to MAI was 10–60% that of the isolated protein mixture. This suggests that the large subunit has a denser structure than the small one.In agreement with earlier experiments with iodoacetamide the proteins S2, 5, 7, 8, 10 and 13 of the small subunit and L15, 17, 20, 24, 25, 27, 29, 33, 34, 35 and 38 in the large subunit are quite accessible while proteins S9, 14, 19, 20, 24, 25, 27, 29 and 30 of the small subunit and L1, 7, 8, 10, 11, 19, 28, 31 and 32 of the large one are relatively inaccessible.     

Isolation and Characterization of Photosystem Ⅱ of Porphyra yezoensis Ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem Ⅱ (PSⅡ) particles were isolated and purified. The activity of PSⅡ     

Identification of 12 new yeast mitochondrial ribosomal proteins including 6 that have no prokaryotic homologues

Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit. Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit. Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein. Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe. In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans. Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Protein Methylation in Cerebellar Synaptosomes

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21–26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21–26-kDa family was identified as GTP-binding proteins by [α-³²P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [α-³²P]GTP. When synaptosomes were screened for putative methyltransferases by S -adenosyl-L-[ methyl -³H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.     

Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum     

A 110-kilodalton subunit of translation initiation factor eIF3 and an associated 135-kilodalton protein are encoded by the Saccharomyces cerevisiae TIF32 and TIF31 genes.

Translation initiation factor eIF3 is a multisubunit protein complex required for initiation of protein biosynthesis in eukaryotic cells. The complex promotes ribosome dissociation, the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit, and mRNA recruitment to the ribosome. In the yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in purified eIF3, we cloned the TIF31 and TIF32 genes encoding 135- (p135) and 110-kDa (p110) proteins. Deletion/disruption of TIF31 results in no change in growth rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of inhibition of the initiation phase. In addition, p110 depletion leads to p90 co-depletion, whereas other eIF3 subunit levels are not affected. Immunoprecipitation or nickel affinity chromatography from strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3 as well as p110 and p33. This establishes p110 as an authentic subunit of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses detect a direct interaction of p110 with p90, p135 with p33, and p33 with eIF4B. Our results, together with those from other laboratories, complete the cloning and characterization of all of the yeast eIF3 subunits.     

Isolation and partial characterization of a glial hyaluronate-binding protein

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.     

Asymmetric interactions between the acidic P1 and P2 proteins in the Saccharomyces cerevisiae ribosomal stalk.

The Saccharomyces cerevisiae ribosomal stalk is made of five components, the 32-kDa P0 and four 12-kDa acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. The P0 carboxyl-terminal domain is involved in the interaction with the acidic proteins and resembles their structure. Protein chimeras were constructed in which the last 112 amino acids of P0 were replaced by the sequence of each acidic protein, yielding four fusion proteins, P0-1alpha, P0-1beta, P0-2alpha, and P0-2beta. The chimeras were expressed in P0 conditional null mutant strains in which wild-type P0 is not present. In S. cerevisiae D4567, which is totally deprived of acidic proteins, the four fusion proteins can replace the wild-type P0 with little effect on cell growth. In other genetic backgrounds, the chimeras either reduce or increase cell growth because of their effect on the ribosomal stalk composition. An analysis of the stalk proteins showed that each P0 chimera is able to strongly interact with only one acidic protein. The following associations were found: P0-1alpha.P2beta, P0-1beta.P2alpha, P0-2alpha.P1beta, and P0-2beta.P1alpha. These results indicate that the four acidic proteins do not form dimers in the yeast ribosomal stalk but interact with each other forming two specific associations, P1alpha.P2beta and P1beta.P2alpha, which have different structural and functional roles.     

Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.     

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.     

Regulated heterogeneity in 12-kDa P-protein phosphorylation and composition of ribosomes in maize (Zea mays L.)

Maize (Zea mays L.) possesses four distinct approximately 12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.