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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Dataset's files

Source:

			Action	DRS
	MSZ08-1_Insol_IGBRP_20150508_1_A1_01_1856.baf	Other
	MSZ08-1_Insol_IGBRP_20150508_2_A2_01_1857.baf	Other
	MSZ08-1_Insol_IGBRP_20150508_3_A3_01_1858.baf	Other
	MSZ08-1_Insol_IGBRP_20150508_4_A4_01_1859.baf	Other
	MSZ08-1_Insol_IGBRP_20150508_5_A5_01_1860.baf	Other

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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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