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Methods (San Diego, Calif.) Mar 2007Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active...
Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histones associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gcn5 subcomplex of SAGA which acetylates histones H3 and H2B on nucleosomes, and the Piccolo NuA4 complex which acetylates histones H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify Escherichia coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes.
Topics: Acetylation; Chromatin Assembly and Disassembly; Chromatography, Affinity; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Fungal; Histone Acetyltransferases; Histones; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Transcription Factors; Yeasts
PubMed: 17309836
DOI: 10.1016/j.ymeth.2006.08.007 -
The Biochemical Journal Jul 19761. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70...
1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.
Topics: Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Ferritins; Hot Temperature; Humans; Immune Sera; Iron; Isoelectric Focusing; Isoelectric Point; Liver; Myocardium; Spleen
PubMed: 962866
DOI: 10.1042/bj1570097 -
Cell Research Dec 2017Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe,...
Telomeric shelterin complex caps chromosome ends and plays a crucial role in telomere maintenance and protection. In the fission yeast Schizosaccharomyces pombe, shelterin is composed of telomeric single- and double-stranded DNA-binding protein subcomplexes Pot1-Tpz1 and Taz1-Rap1, which are bridged by their interacting protein Poz1. However, the structure of Poz1 and how Poz1 functions as an interaction hub in the shelterin complex remain unclear. Here we report the crystal structure of Poz1 in complex with Poz1-binding motifs of Tpz1 and Rap1. The crystal structure shows that Poz1 employs two different binding surfaces to interact with Tpz1 and Rap1. Unexpectedly, the structure also reveals that Poz1 adopts a dimeric conformation. Mutational analyses suggest that proper interactions between Tpz1, Poz1, and Rap1 in the shelterin core complex are required for telomere length homeostasis and heterochromatin structure maintenance at telomeres. Structural resemblance between Poz1 and the TRFH domains of other shelterin proteins in fission yeast and humans suggests a model for the evolution of shelterin proteins.
Topics: Carrier Proteins; DNA-Binding Proteins; Protein Conformation; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Shelterin Complex; Telomere; Telomere-Binding Proteins
PubMed: 29160296
DOI: 10.1038/cr.2017.145 -
The Journal of Biological Chemistry May 1996Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids....
Purification, identification, and properties of a Saccharomyces cerevisiae oleate-activated upstream activating sequence-binding protein that is involved in the activation of POX1.
Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the beta-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480-24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.
Topics: Acyl-CoA Oxidase; Amino Acid Sequence; Base Sequence; DNA-Binding Proteins; Enzyme Activation; Fungal Proteins; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Oleic Acid; Oleic Acids; Oxidoreductases; Promoter Regions, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors
PubMed: 8662598
DOI: 10.1074/jbc.271.20.12068 -
Scientific Reports May 2017During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal...
During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal complex (SC) in other organisms. LinE proteins also promote the formation of meiotic DNA double-strand breaks (DSBs), the precursors of cross-overs. Rec10 is required for essentially all DSBs and recombination, and three others (Rec25, Rec27, and Mug20) are protein determinants of DSB hotspots - they bind DSB hotspots with high specificity and are required for DSB formation there. These four LinE proteins co-localize in the nucleus in an interdependent way, suggesting they form a complex. We used random mutagenesis to uncover recombination-deficient missense mutants with novel properties. Some missense mutations changed essential residues conserved among Schizosaccharomyces species. DSB formation, gene conversion, and crossing-over were coordinately reduced in the mutants tested. Based on our mutant analysis, we revised the rec27 open reading frame: the new start codon is in the previously annotated first intron. Genetic and fluorescence-microscopy assays indicated that the Rec10 N- and C-terminal regions have complex interactions with Rec25. These mutants are a valuable resource to elucidate further how LinE proteins and the related SCs of other species regulate meiotic DSB formation to form crossovers crucial for meiosis.
Topics: Cell Cycle Proteins; DNA Breaks, Double-Stranded; Gene Conversion; Introns; Meiosis; Mutation, Missense; Nuclear Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 28469148
DOI: 10.1038/s41598-017-00742-3 -
Molecular and Cellular Biology Oct 2002To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors,...
To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.
Topics: Amino Acid Sequence; Carrier Proteins; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Fungal Proteins; Histone Chaperones; Molecular Sequence Data; Nuclear Proteins; Peptide Elongation Factors; Phosphoproteins; Proteome; RNA Polymerase II; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcription Factors, General; Transcription Factors, TFII; Transcriptional Elongation Factors
PubMed: 12242279
DOI: 10.1128/MCB.22.20.6979-6992.2002 -
The Journal of Biological Chemistry Jan 1994C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP...
C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP complex is anticoagulantly active. To identify beta chain residues responsible for binding protein S, synthetic overlapping pentadecapeptides covering the entire 1-235 sequence were tested as inhibitors of complex formation. The peptide comprising residues 31-45 (VCIKGYHLVGKKTLF) from the first short consensus repeat domain inhibited the binding of C4BP to protein S with half-maximal inhibition at 20-45 microM, and studies suggested the sequence of YxLVG was crucial. Peptide beta(31-45) specifically inhibited the APC cofactor activity of purified protein S in Xa-1-stage coagulation assays with 50% inhibition at 15 microM peptide. Peptide beta(31-45) and related peptides such as beta(34-42) inhibited the binding of protein S to an antipeptide monoclonal antibody made against residues 420-434 of protein S (monoclonal antibody LJ-56). Polyclonal anti-beta(31-45) peptide antibodies inhibited complex formation. Dose-dependent binding studies showed that protein S bound directly to immobilized peptide beta(31-45). These results show that residues 31-45 of the C4BP beta chain provide a binding site for protein S, and they suggest that the C4BP beta chain residues 34-42 are located near residues 420-434 of protein S in the protein S:C4BP complex.
Topics: Amino Acid Sequence; Binding Sites; Blood Coagulation; Carrier Proteins; Complement C4b; Complement Inactivator Proteins; Consensus Sequence; Glycoproteins; Humans; Kinetics; Macromolecular Substances; Molecular Sequence Data; Peptide Fragments; Peptides; Protein S; Structure-Activity Relationship
PubMed: 8300581
DOI: No ID Found -
Nature Communications Dec 2019Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1,...
Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.
Topics: Crystallography, X-Ray; Nucleocytoplasmic Transport Proteins; Proteasome Endopeptidase Complex; Protein Binding; Protein Interaction Domains and Motifs; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitination; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 31836717
DOI: 10.1038/s41467-019-13697-y -
Analytical Chemistry Aug 2010Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically...
Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.
Topics: Buffers; Cell Cycle Proteins; Cell Line; Histidine; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Mechanistic Target of Rapamycin Complex 1; Microtubule-Associated Proteins; Multiprotein Complexes; Oligopeptides; Peptides; Phosphorylation; Protein Isoforms; Protein Subunits; Proteins; Saccharomyces cerevisiae Proteins; TOR Serine-Threonine Kinases; Temperature; Transcription Factors; Viscosity
PubMed: 20614870
DOI: 10.1021/ac101235k -
The EMBO Journal Nov 1993SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a...
SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13-dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate-agarose affinity chromatography facilitated the purification of two forms of the Sec13-dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13-dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell-free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13-dhfrp/p150 complex, but not the Sec13-dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell-free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.
Topics: Biological Transport; Cell-Free System; Cells, Cultured; Cytosol; Endoplasmic Reticulum; Fungal Proteins; Golgi Apparatus; Macromolecular Substances; Membrane Fusion; Membrane Proteins; Nuclear Pore Complex Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins; Tetrahydrofolate Dehydrogenase
PubMed: 8223424
DOI: 10.1002/j.1460-2075.1993.tb06091.x