Dataset Information

Yeast Heatshock SILAC LC-MSMS

ABSTRACT: Yeast cells were incubated at 25˚C, unless otherwise stated. For SILAC, cells were grown in light labeled (0.03mg/ml Lys0, 0.02mg/ml Arg0) or heavy labeled (0.03mg/ml Lys4, 0.02mg/ml Arg6) amino acid in synthetic defined medium with 2% dextrose for at least 7 generations. heat shock was performed in a shaking water bath at 45˚C for 20 minutes before harvest. Equal OD600 of cells from both labels were collected at mid log phase (OD¬600=0.8-1) by centrifugation at 3,220xg for 5 minutes at 4˚C, washed twice with cold 1×TBS (50mM Tris pH 7.5, 150mM NaCl), mixed, and re-suspended in equal volume of 2×Native lysis buffer (200mM Tris pH 7.5, 150mM NaCl, 2mM PMSF, 2×Protease Inhibitor Cocktails (Sigma-Aldrich), 2mM 1,10-Phenanthroline). Re-suspended cells were snap-frozen drop by drop in liquid nitrogen. The resulting cells pellets were lysed by cyro-grinding in liquid nitrogen with a mortar and pestle mounted on an electric driver (OPS Diagnostics). The lysate was thawed on ice and further diluted 3 times by adding ice-cold 1×Native lysis buffer and 1% NP40 (final concentration). Total cell lysate was pre-cleared twice by centrifugation at 1,000×g at 4˚C for 15min. Pellet fraction was separated by centrifugation at 16,100×g at 4˚C for 15min. The insoluble protein pellet was washed twice with ice-cold 1×Native lysis buffer containing 1% NP40 and re-solubilized in 1×Laemmli Sample Buffer (62.5mM Tris-HCl pH6.8, 2% SDS, 10% Glycerol). Protein concentrations were determined by using BioRad DC™ Protein Assay.

INSTRUMENT(S): impact II

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Mang Zhu

LAB HEAD: Thibault Mayor

PROVIDER: PXD012631 | Pride | 2020-06-12

REPOSITORIES: Pride

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Publications

Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in <i>S. cerevisiae</i>.

Zhu Mang M Kuechler Erich R ER Zhang Joyce J Matalon Or O Dubreuil Benjamin B Hofmann Analise A Loewen Chris C Levy Emmanuel D ED Gsponer Joerg J Mayor Thibault T

Journal of cell science 20200709 13

Stress granules (SGs) are stress-induced membraneless condensates that store non-translating mRNA and stalled translation initiation complexes. Although metazoan SGs are dynamic compartments where proteins can rapidly exchange with their surroundings, yeast SGs seem largely static. To gain a better understanding of yeast SGs, we identified proteins that sediment after heat shock using mass spectrometry. Proteins that sediment upon heat shock are biased toward a subset of abundant proteins that a ...[more]

PMID: 32503941

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Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

Dataset Information

Yeast Heatshock SILAC LC-MSMS

Publications

Proteomic analysis reveals the direct recruitment of intrinsically disordered regions to stress granules in <i>S. cerevisiae</i>.

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