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The Journal of Biological Chemistry Dec 2009Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins...
Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins (mRNPs)) before export to the cytoplasm. Here, we have used the poly(A)-binding protein Nab2 to isolate mRNPs from yeast under conditions that preserve mRNA integrity. Upon Nab2-tandem affinity purification, several mRNA export factors were co-enriched (Yra1, Mex67, THO-TREX) that were present in mRNPs of different size and mRNA length. High-throughput sequencing of the co-precipitated RNAs indicated that Nab2 is associated with the bulk of yeast transcripts with no specificity for different mRNA classes. Electron microscopy revealed that many of the mRNPs have a characteristic elongated structure. Our data suggest that mRNPs, although associated with different mRNAs, have a unifying core structure.
Topics: Gene Expression Profiling; Nucleic Acid Conformation; Nucleocytoplasmic Transport Proteins; Protein Binding; Protein Conformation; RNA, Fungal; RNA, Messenger; RNA-Binding Proteins; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 19840948
DOI: 10.1074/jbc.M109.062034 -
Journal of Biochemistry May 1985A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically...
A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z.
A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).
Topics: Amino Acids; Animals; Blood Coagulation Factors; Blood Proteins; Cattle; Chromatography; Factor IX; Factor X; Glycoproteins; Molecular Weight; Protein C; Protein S; Prothrombin; Vitamin K
PubMed: 3161875
DOI: 10.1093/oxfordjournals.jbchem.a135187 -
The EMBO Journal Apr 2018Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S...
Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.
Topics: Cryoelectron Microscopy; Cytoplasm; Molecular Docking Simulation; Nuclear Proteins; Protein Conformation; Protein Domains; Protein Interaction Domains and Motifs; Protein Serine-Threonine Kinases; RNA Folding; RNA, Ribosomal; RNA-Binding Proteins; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 29459436
DOI: 10.15252/embj.201798499 -
DNA Repair Sep 2013The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of...
The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.
Topics: Adenosine Triphosphate; Calcium; Cell Cycle Proteins; Chromatography, Gel; DNA Helicases; DNA Repair Enzymes; DNA Topoisomerases; DNA, Fungal; DNA, Single-Stranded; DNA-Binding Proteins; Homologous Recombination; Humans; Hydrolysis; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 23769192
DOI: 10.1016/j.dnarep.2013.05.004 -
Protein Science : a Publication of the... Dec 2018RWD domains mediate protein-protein interactions in a variety of pathways in eukaryotes. In budding yeast, the RWD domain protein Csm1 is particularly versatile,...
RWD domains mediate protein-protein interactions in a variety of pathways in eukaryotes. In budding yeast, the RWD domain protein Csm1 is particularly versatile, assembling key complexes in the nucleolus and at meiotic kinetochores through multiple protein interaction surfaces. Here, we reveal a third functional context for Csm1 by identifying a new Csm1-interacting protein, Dse3. We show that Dse3 interacts with Csm1 in a structurally equivalent manner to its known binding partners Mam1 and Ulp2, despite these three proteins' lack of overall sequence homology. We theorize that the unique "clamp" structure of Csm1 and the loose sequence requirements for Csm1 binding have led to its incorporation into at least three different structural/signaling pathways in budding yeast.
Topics: Cell Cycle Proteins; Crystallography, X-Ray; Models, Molecular; Nuclear Proteins; Protein Binding; Protein Conformation; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 30252178
DOI: 10.1002/pro.3515 -
The Journal of Biological Chemistry Jan 1990We have purified extensively the transcriptional activator, GAL4, from a yeast strain overexpressing the gene product from the ADH1 promoter. Our purification followed...
We have purified extensively the transcriptional activator, GAL4, from a yeast strain overexpressing the gene product from the ADH1 promoter. Our purification followed GAL4 activity by its binding to a specific DNA target sequence, using filter binding assays. No specific binding activity was detected in extracts from a strain containing a disrupted copy of the GAL4 gene. The purification protocol included fractionation of a whole cell extract by ion-exchange and DNA-affinity chromatography on a column containing a 17-base pair oligomer encoding a near consensus GAL4 binding site. Two polypeptides co-eluted with the GAL4 DNA binding activity from the DNA-affinity column. One had an apparent molecular mass of 99 kDa (the predicted size of the GAL4 protein) and cross-reacted with antibodies raised against GAL4 epitopes from fusion proteins expressed in bacterial cells. The second polypeptide did not cross-react with the anti-GAL4 antibody and is presumed to be the GAL80 transcriptional repressor based on its size (48 kDa) and known physical association with the GAL4 protein. GAL4 binding activity elutes from a gel filtration column as a 155-kDa species suggesting that it exists in solution in a heterodimer complex of one GAL4 and one GAL80 molecule. The dissociation constant of the DNA-affinity-purified GAL4-GAL80 complex for a 900-base pair DNA fragment containing the UASGAL element from the GAL1-GAL10 divergent promoter was, Kd(effective) (0.15 M KCl) = 2.4 x 10(-9) M.
Topics: Blotting, Western; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; DNA; DNA-Binding Proteins; Fungal Proteins; Macromolecular Substances; Molecular Weight; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors
PubMed: 2403556
DOI: No ID Found -
Protein Expression and Purification Jul 2013Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a...
Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.
Topics: Chromatography, Gel; Detergents; Electrophoresis, Polyacrylamide Gel; HIV Envelope Protein gp120; HIV Envelope Protein gp160; Organic Chemicals; Protein Precursors; Recombinant Proteins; Vaccinia virus
PubMed: 23665667
DOI: 10.1016/j.pep.2013.04.009 -
Proceedings of the National Academy of... Jun 1983C4b-binding protein (C4bp) participates in the regulation of the C3 convertase of the classical pathway of complement. By binding to C4b, which is one of the structural...
C4b-binding protein (C4bp) participates in the regulation of the C3 convertase of the classical pathway of complement. By binding to C4b, which is one of the structural subunits of this enzyme, C4bp accelerates the decay-dissociation of the enzyme and renders C4b susceptible to degradation by factor I (C3b inactivator). C4bp is a high molecular weight plasma protein (Mr = 570,000) composed of apparently identical subunits (Mr = 70,000) linked by disulfide bonds. In plasma and in purified form C4bp also forms a bimolecular complex (Kd = 0.9 X 10(-7) M) with protein S, a recently identified vitamin K-dependent plasma protein. The binding sites on C4bp for protein S and C4b are distinct and noncompetitive and protein S does not influence the function of C4bp as a regulator of the C3 convertase. C4bp, C4b, and protein S were visualized by electron microscopy by negative staining. C4bp was found to have an unusual spider-like structure. It is composed of seven thin (30 A), elongated (330 A), and flexible subunits that are linked to a small central body. Protein S exhibited two globular domains of equal size with a center-to-center distance of approximately equal to 50 A. Protein S was found to bind to the C4bp through only one of its domains by attaching to a short subunit that is distinct from the other seven subunits. C4b imaged as an irregular, relatively compact molecule. It was found to interact with the peripheral ends of the elongated subunits, suggesting seven C4b-binding sites per molecule of C4bp.
Topics: Carrier Proteins; Complement C4; Complement C4b; Complement Inactivator Proteins; Glycoproteins; Humans; Macromolecular Substances; Microscopy, Electron; Molecular Weight; Protein S
PubMed: 6222381
DOI: 10.1073/pnas.80.11.3461 -
Molecular & Cellular Proteomics : MCP Jun 2014The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new...
The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.
Topics: Cytoplasm; Gene Expression Regulation; HEK293 Cells; Humans; Mass Spectrometry; Multiprotein Complexes; Protein Interaction Maps; Proteomics; Transcription Factor RelA; Transcription Factors
PubMed: 24722732
DOI: 10.1074/mcp.M113.033902 -
Molecular and Cellular Biology Mar 2008Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human...
Rad23 is required for efficient protein degradation and performs an important role in nucleotide excision repair. Saccharomyces cerevisiae Rad23, and its human counterpart (hHR23), are present in a complex containing the DNA repair factor Rad4 (termed XPC, for xeroderma pigmentosum group C, in humans). XPC/hHR23 was also reported to bind centrin-2, a member of the superfamily of calcium-binding EF-hand proteins. We report here that yeast centrin, which is encoded by CDC31, is similarly present in a complex with Rad4/Rad23 (called NEF2). The interaction between Cdc31 and Rad23/Rad4 varied by growth phase and reflected oscillations in Cdc31 levels. Strikingly, a cdc31 mutant that formed a weaker interaction with Rad4 showed sensitivity to UV light. Based on the dual function of Rad23, in both DNA repair and protein degradation, we questioned if Cdc31 also participated in protein degradation. We report here that Cdc31 binds the proteasome and multiubiquitinated proteins through its carboxy-terminal EF-hand motifs. Moreover, cdc31 mutants were highly sensitive to drugs that cause protein damage, failed to efficiently degrade proteolytic substrates, and formed altered interactions with the proteasome. These findings reveal for the first time a new role for centrin/Cdc31 in protein degradation.
Topics: Adenosine Triphosphatases; Calcium-Binding Proteins; Cell Cycle Proteins; Cloning, Molecular; Cycloheximide; DNA Damage; DNA, Fungal; DNA-Binding Proteins; Escherichia coli; Gene Amplification; Genes, Essential; Genes, Fungal; Glutathione Transferase; Hygromycin B; Mutation; Plasmids; Proteasome Endopeptidase Complex; Protein Binding; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitins; Ultraviolet Rays
PubMed: 18160718
DOI: 10.1128/MCB.01256-07