-
The Journal of Biological Chemistry Jan 1991Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two...
Transcription of the GAL genes encoding the enzymes responsible for galactose metabolism in the yeast Saccharomyces cerevisiae is regulated through an interplay of two regulatory proteins, GAL4 and GAL80. GAL4 binds to upstream activating sequences of GAL (UASG) and activates their transcription in yeast growing in the presence of galactose. GAL80 binds to GAL4 and inhibits the activation function of GAL4 in yeast growing without galactose. We have purified GAL80 in its native form as a protein that reacts with an antiserum raised against a synthetic peptide of 18 amino acid residues in the GAL80 sequence. Purification was performed through ammonium sulfate precipitation, streptomycin precipitation, DEAE-cellulose column chromatography, and gel filtration. From 50 g of wet cells, a final sample of 2.3 mg with a purity of more than 80% was obtained. The molecular size of the purified protein in both the native and denatured states was estimated to be approximately 50 kDa, indicating that GAL80 exists as a monomer in yeast cells. The amino-terminal residue of GAL80 was found to be acetylmethionine. The purified protein was shown to bind GAL4. We have also purified mutant GAL80 proteins encoded by two different alleles of gal80 known to be incapable of inhibiting the function of GAL4. These proteins were, in fact, unable to bind GAL4.
Topics: Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; Molecular Weight; Mutation; Peptides; Repressor Proteins; Saccharomyces cerevisiae Proteins
PubMed: 1985957
DOI: No ID Found -
The Plant Journal : For Cell and... Aug 1994Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast...
Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae.
Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [3H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of D-glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-beta-D-glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni(2+)-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.
Topics: Amino Acid Sequence; Arabidopsis; Base Sequence; Carrier Proteins; DNA, Plant; Gene Expression; Histidine; Hydrogen; Liposomes; Molecular Sequence Data; Monosaccharides; Nuclear Proteins; Plant Proteins; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Tagged Sites; Solubility; Transcription Factors
PubMed: 7920712
DOI: 10.1046/j.1365-313x.1994.6020225.x -
Molecular & Cellular Proteomics : MCP Feb 2012Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their...
Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions.
Topics: Animals; Brain; Cell Membrane; Chromatography, Affinity; Chromatography, Liquid; Fourier Analysis; Kv1.1 Potassium Channel; Mice; Proteomics; Rats; Tandem Mass Spectrometry
PubMed: 22067099
DOI: 10.1074/mcp.M111.007955 -
Current Protocols in Cell Biology Jun 2015In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently...
In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro.
Topics: Biological Assay; Electrophoresis, Polyacrylamide Gel; Peptide Hydrolases; Polyubiquitin; Powders; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Ubiquitinated Proteins
PubMed: 26061243
DOI: 10.1002/0471143030.cb0343s67 -
Genetics May 2009The completion of genome-sequencing projects for a number of fungi set the stage for detailed investigations of proteins. We report the generation of versatile...
The completion of genome-sequencing projects for a number of fungi set the stage for detailed investigations of proteins. We report the generation of versatile expression vectors for detection and isolation of proteins and protein complexes in the filamentous fungus Neurospora crassa. The vectors, which can be adapted for other fungi, contain C- or N-terminal FLAG, HA, Myc, GFP, or HAT-FLAG epitope tags with a flexible poly-glycine linker and include sequences for targeting to the his-3 locus in Neurospora. To introduce mutations at native loci, we also made a series of knock-in vectors containing epitope tags followed by the selectable marker hph (resulting in hygromycin resistance) flanked by two loxP sites. We adapted the Cre/loxP system for Neurospora, allowing the selectable marker hph to be excised by introduction of Cre recombinase into a strain containing a knock-in cassette. Additionally, a protein purification method was developed on the basis of the HAT-FLAG tandem affinity tag system, which was used to purify HETEROCHROMATIN PROTEIN 1 (HP1) and associated proteins from Neurospora. As expected on the basis of yeast two-hybrid and co-immunoprecipitation (Co-IP) experiments, the Neurospora DNA methyltransferase DIM-2 was found in a complex with HP1. Features of the new vectors allowed for verification of an interaction between HP1 and DIM-2 in vivo by Co-IP assays on proteins expressed either from their native loci or from the his-3 locus.
Topics: Chromatography, Affinity; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Fungal Proteins; Gene Transfer Techniques; Genetic Vectors; Immunoprecipitation; Integrases; Molecular Sequence Data; Neurospora crassa; Plasmids; Proteomics; Saccharomyces cerevisiae Proteins
PubMed: 19171944
DOI: 10.1534/genetics.108.098707 -
Acta Crystallographica. Section F,... May 2013The membrane protein Erv41p is a major component of COPII-coated vesicles and is thought to play a role in the early secretory pathway in eukaryotic cells. In this...
The membrane protein Erv41p is a major component of COPII-coated vesicles and is thought to play a role in the early secretory pathway in eukaryotic cells. In this study, the full lumenal domain of Erv41p from Saccharomyces cerevisiae (ScErv41p_LD) was recombinantly expressed in Sf9 insect cells and purified by nickel-affinity, ion-exchange and size-exclusion chromatography. ScErv41p_LD crystals were obtained using the sitting-drop vapour-diffusion method and native X-ray diffraction data were collected to 2.0 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 49.8, b = 76.9, c = 65.1 Å, α = γ = 90.0, β = 104.8°.
Topics: Crystallization; Crystallography, X-Ray; Endoplasmic Reticulum; Membrane Proteins; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; X-Ray Diffraction
PubMed: 23695573
DOI: 10.1107/S1744309113008063 -
Nucleic Acids Research Jul 2019Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes.... (Comparative Study)
Comparative Study
Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.
Topics: Amino Acid Sequence; Models, Molecular; Molecular Chaperones; Nucleosome Assembly Protein 1; Organelle Biogenesis; Protein Binding; Protein Biosynthesis; Protein Conformation; Protein Domains; Protein Interaction Mapping; Recombinant Fusion Proteins; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 31062022
DOI: 10.1093/nar/gkz317 -
Nucleic Acids Research 2006SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and...
SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure-function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5-Slx8 may act directly on DNA to promote genome stability.
Topics: DNA; DNA-Binding Proteins; Protein Structure, Tertiary; Protein Subunits; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Substrate Specificity; Ubiquitin-Protein Ligases
PubMed: 17020915
DOI: 10.1093/nar/gkl685 -
European Journal of Biochemistry Oct 1997Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear... (Comparative Study)
Comparative Study
Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.
Topics: Amino Acid Sequence; Animals; Caenorhabditis elegans; Carrier Proteins; Chickens; Fungal Proteins; Humans; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Nucleic Acids; Ploidies; Protein Binding; Proteins; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Species Specificity; Subcellular Fractions
PubMed: 9363784
DOI: 10.1111/j.1432-1033.1997.00309.x -
The Biochemical Journal Jun 1991Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family...
Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.
Topics: Amino Acid Sequence; Chromatography; Enzyme Activation; Fungal Proteins; GTP Phosphohydrolases; GTP-Binding Proteins; GTPase-Activating Proteins; Humans; Membrane Proteins; Molecular Sequence Data; Proteins; Repressor Proteins; Saccharomyces cerevisiae Proteins; Sequence Homology, Nucleic Acid; Spleen; ras GTPase-Activating Proteins; rhoB GTP-Binding Protein
PubMed: 1905930
DOI: 10.1042/bj2760833