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Nature Communications Sep 2021In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70...
In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.
Topics: Adenosine Triphosphate; HSP70 Heat-Shock Proteins; Hydrolysis; Molecular Chaperones; Molecular Docking Simulation; Protein Domains; Protein Folding; Protein Multimerization; Recombinant Proteins; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tandem Mass Spectrometry
PubMed: 34580293
DOI: 10.1038/s41467-021-25930-8 -
Molecular and Cellular Biology Sep 1993The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and...
The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.
Topics: Allosteric Regulation; Base Sequence; Binding Sites; Cell Cycle Proteins; DNA-Binding Proteins; Fungal Proteins; Genes, Fungal; Homeodomain Proteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Trans-Activators; Transcription Factors; Zinc Fingers
PubMed: 8355698
DOI: 10.1128/mcb.13.9.5524-5537.1993 -
Methods (San Diego, Calif.) Dec 2011Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that... (Review)
Review
Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.
Topics: Amino Acid Sequence; Bacterial Proteins; Chromatography, Liquid; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Multiprotein Complexes; Plant Proteins; Protein Processing, Post-Translational; Tandem Mass Spectrometry
PubMed: 21982782
DOI: 10.1016/j.ymeth.2011.09.019 -
Nature Communications Sep 2021Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of...
Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.
Topics: Cell Fractionation; Cell Nucleus; Cross-Linking Reagents; Mass Spectrometry; Membrane Transport Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Molecular Docking Simulation; Mutagenesis, Site-Directed; Point Mutation; Protein Binding; Protein Interaction Mapping; Protein Precursors; Recombinant Proteins; Saccharomyces cerevisiae Proteins
PubMed: 34588454
DOI: 10.1038/s41467-021-26016-1 -
Methods in Molecular Biology (Clifton,... 2019Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means...
Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.
Topics: Adenosine Triphosphatases; Liposomes; Membrane Fusion; Phospholipids; Protein Binding; SNARE Proteins; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 30317507
DOI: 10.1007/978-1-4939-8760-3_13 -
Methods in Molecular Biology (Clifton,... 2018The addition of nitric oxide to cysteine moieties of proteins results in the formation of S-nitrosothiols (SNO) that have emerged as important posttranslational...
The addition of nitric oxide to cysteine moieties of proteins results in the formation of S-nitrosothiols (SNO) that have emerged as important posttranslational signaling cues in a wide variety of eukaryotic processes. While formation of protein-SNO is largely nonenzymatic, the conserved family of Thioredoxin (TRX) enzymes are capable of selectively reducing protein-SNO. Consequently, TRX enzymes are thought to provide reversibility and specificity to protein-SNO signaling networks. Here, we describe an in vitro methodology based on enzymatic oxidoreductase and biotin-switch techniques, allowing for the detection of protein-SNO targets of TRX enzymes. We show that this methodology identifies both global and specific protein-SNO targets of TRX in plant cell extracts.
Topics: Biotin; Biotinylation; Nitric Oxide; Nitrosation; Plants; Protein Processing, Post-Translational; Recombinant Proteins; S-Nitrosothiols; Thioredoxins
PubMed: 29600467
DOI: 10.1007/978-1-4939-7695-9_22 -
Proceedings of the National Academy of... Oct 1994Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of...
Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of digitonin-permeabilized cells. Fraction A is required for recognition of the nuclear localization sequence and targeting to the nuclear envelope, whereas fraction B is required for the subsequent translocation of the bound substrate into the nucleus. The first protein required for fraction B activity to be purified was the small GTPase Ran (ras-related nuclear protein). Here we report the purification of the second (and final) protein required for fraction B activity. By SDS/PAGE, the purified protein appeared as a single band with an apparent molecular mass of 10 kDa, but the native protein fractionated upon gel filtration chromatography with an apparent size of 30 kDa. Peptide sequence analysis indicated that the purified protein was highly homologous to a previously identified human protein of unknown function called placental protein 15 (pp15) and to the predicted protein product of a yeast open reading frame from Saccharomyces cerevisiae.
Topics: Amino Acid Sequence; Animals; Carrier Proteins; Cell Nucleus; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Cytosol; Female; GTP-Binding Proteins; Kinetics; Liver; Molecular Sequence Data; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Ovary; Rats; Rats, Inbred BUF; Saccharomyces cerevisiae Proteins; Xenopus Proteins; Xenopus laevis; ran GTP-Binding Protein
PubMed: 7937864
DOI: 10.1073/pnas.91.21.10212 -
Cell Stress & Chaperones Sep 1999Alcoholic extracts of bakers' yeast (Saccharomyces cerevisiae) have been used for over 60 years in over-the-counter medications for the treatment of hemorrhoids, burns,...
Alcoholic extracts of bakers' yeast (Saccharomyces cerevisiae) have been used for over 60 years in over-the-counter medications for the treatment of hemorrhoids, burns, and wounds. Although previous studies suggested that small peptides were responsible for the medical observations, the peptides were never resolved into separate fractions and identified. In the present report, a protein fraction was prepared by RPC18 chromatography of the extract which enhances wound closure in both diabetic and non-diabetic littermates. The peptides are active in nanomolar amounts and are 600 times more active than the initial extract. SDS-PAGE and N-terminal amino acid sequencing identified 4 polypeptides in the extract. Three of the proteins were small molecular weight stress-associated proteins: copper, zinc superoxide-dismutase, ubiquitin, and glucose lipid regulated protein (HSP 12). The fourth protein, acyl-CoA binding protein II, has not been previously associated with stress proteins.
Topics: Amino Acid Sequence; Carrier Proteins; Diazepam Binding Inhibitor; Free Radical Scavengers; Fungal Proteins; Heat-Shock Proteins; Humans; Molecular Sequence Data; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Superoxide Dismutase; Ubiquitins; Wounds and Injuries
PubMed: 10547066
DOI: 10.1379/1466-1268(1999)004<0171:mye>2.3.co;2 -
Applied and Environmental Microbiology Dec 2000The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain...
The yeast Schwanniomyces occidentalis produces a killer toxin lethal to sensitive strains of Saccharomyces cerevisiae. Killer activity is lost after pepsin and papain treatment, suggesting that the toxin is a protein. We purified the killer protein and found that it was composed of two subunits with molecular masses of approximately 7.4 and 4.9 kDa, respectively, but was not detectable with periodic acid-Schiff staining. A BLAST search revealed that residues 3 to 14 of the 4.9-kDa subunit had 75% identity and 83% similarity with killer toxin K2 from S. cerevisiae at positions 271 to 283. Maximum killer activity was between pH 4.2 and 4.8. The protein was stable between pH 2.0 and 5.0 and inactivated at temperatures above 40 degrees C. The killer protein was chromosomally encoded. Mannan, but not beta-glucan or laminarin, prevented sensitive yeast cells from being killed by the killer protein, suggesting that mannan may bind to the killer protein. Identification and characterization of a killer strain of S. occidentalis may help reduce the risk of contamination by undesirable yeast strains during commercial fermentations.
Topics: Amino Acid Sequence; Binding, Competitive; Fermentation; Food Technology; Fungal Proteins; Hydrogen-Ion Concentration; Killer Factors, Yeast; Molecular Sequence Data; Molecular Weight; Mycotoxins; Phenotype; Plasmids; Polysaccharides; Protein Subunits; Saccharomyces cerevisiae Proteins; Saccharomycetales
PubMed: 11097913
DOI: 10.1128/AEM.66.12.5348-5352.2000 -
The Biochemical Journal Mar 1983C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps....
C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.
Topics: Amino Acids; Carrier Proteins; Complement Inactivator Proteins; Electrophoresis, Agar Gel; Glycoproteins; Humans; Kinetics; Macromolecular Substances; Molecular Weight; Protein Binding; Protein S; Temperature
PubMed: 6223625
DOI: 10.1042/bj2090847