MassIVE MSV000100570

Complete Public PXD073487

Native yeast kinetochore structures identify an essential inner kinetochore interaction.

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Description

Kinetochores were purified from Saccharomyces cerevisiae (W303) cell lysate after being treated with benzonase and sucrose gradient to remove contaminant proteins. The endogenous AME1 kinetochore gene was C-terminally tagged with 3xM3DK. Harvested yeast were resuspended in Buffer H (25 mM HEPES pH 8.0, 150 mM KCl, 2 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.1% NP-40, 15% glycerol) supplemented with protease inhibitors, phosphatase inhibitors, and 2 mM DTT. After resuspension and re-spinning, yeast pellets were frozen in liquid nitrogen and lysed using a Freezer Mill (SPEX, Metuchen NJ). Lysate was clarified via tabletop centrifugation at maximum speed for 30 minutes. The supernatant was pooled in ultracentrifugation tubes with 3mL sucrose gradient cushion from 25% to 40%. The lysate was ultra spun overnight at 27,000RPM and the clear layer of lysate was extracted with a syringe. This extract was incubated with 625ng/mL 500bp CEN3 DNA at room temperature for 90 minutes and magnetic a-M3DK antibody conjugated Dynabeads (Invitrogen, Waltham MA) were added and incubated for 90 minutes at 4 C with rotation. Dynabeads were washed with Buffer L (25 mM HEPES pH 8.0, 175 mM KGlutamate, 6 mM Mg(OAc)2, 0.1 mM EDTA pH 7.6, 0.5 mM EGTA-KOH, 0.1% NP-40, 15% glycerol, pH7.6) for 5 times (the last 2 washes omitting NP-40, glycerol and phosphatase inhibitors). For mass spectrometry, kinetochores were eluted from Dynabeads with 0.2% RapiGest (Waters Corporation, Milford MA) in 50 mM HEPES pH 8.0. [doi:10.25345/C5VT1H37V] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Mitosis, kinetochore, centromere, cell division ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Susan Biggins, Fred Hutchinson Cancer Center, USA
Submitting User: FHproteomics
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