MassIVE MSV000095817

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A single centromeric nucleosome mediates attachments to a single microtubule in the thermotolerant yeast K. marxianus

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Description

Kinetochores were purified from Kluyveromyces marxianus cell lysate after either being treated with benzonase (DB060424_061124_18150_Benzonase_1, DB060424_061124_18150_Benzonase_2, DB032421_033021_B) or with no treatment (DB060424_061124_18150_control_1, DB060424_061124_18150_control_2, DB032421_033021_C). The endogenous DSN1 kinetochore gene was C-terminally tagged with 6xHis and 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 ultracentrifugation at 24,000 RPM (98,000 x g) for 90 minutes and the protein layer was extracted with a syringe. This extract was incubated with magnetic a-M3DK antibody conjugated Dynabeads (Invirtrogen, Waltham MA) for 3 hours at 4 C with rotation. For benzonase treated samples, 300 units of benzonase per milliliter of lysate were added during this incubation step. Dynabeads were washed with 10x bead volume of Buffer H 5 times (the last 3 washes omitting DTT 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. For all experiments, the total protein concentration was determined by NanoDrop measurement and the relative purity by silver stain gel analysis. [doi:10.25345/C5HQ3S960] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Mitosis, kinetochore, centromere, cell division, Kluyveromyces marxianus

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