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Analysis of a cancer-associated mutation in the budding yeast Nuf2 kinetochore protein

ABSTRACT: Saccharomyces cerevisiae kinetochores were isolated from a NUF2-3V5 background via Dsn1-His-Flag purifications. (AAL020924_02154_SBY23028) and (AAL020924_021524_SBY23066) were grown at 30 C and harvested once cell cultures reached OD600nm = ~3. Cells were resuspended in Buffer H (25mM HEPES pH8.0, 2mM MgCl2 , 0.1 mM EDTA pH 8.0, 0.5 mM EGTA-KOH pH8.0, 15% Glycerol, 0.1% NP-40, and 150 mM KCl) containing phosphatase inhibitors (1 mM sodium-pyrophosphate, 2 mM sodium-beta-glycerophosphate, 0.1 mM sodium orthovanadate, and 5 mM NaF), 0.1 mM microcystin-LR, 0.2 mM PMSF, and protease inhibitors (10 mg/mL for each of the following leupeptin, pepstatin A, and chymostatin). In which cells were spun down and flash frozen in liquid nitrogen. Pellets were lysed via Freezer Mill (SPEX SamplePrep) and treated with 50 units/ml of benzonase (EMD Milipore Corp) for 30 minutes on ice. Lysate proteins were extracted after undergoing ultracentrifugation at 24,000 RPM for 90 minutes at 4 °C. The Pierce BCA Protein Assay Kit (Thermo Scientific) was used to measure the protein concentration within each extract which were subsequently normalized. Extracts were incubated with a-M3DK (GenScript) magnetic Protein G Dynabeads (Invitrogen) for 3 hours at 4 °C with rotation. Beads were washed three times with BH0.15 containing phosphatase inhibitors, protease inhibitors, 0.1 mM microcystin-LR, and 2 mM DTT. Then two times with BH0.15 containing protease inhibitors and LPC. Then rinsed two times with pre-elution rinse buffer (50 mM Tris pH 8.3, 75 mM KCl, and 1 mM EGTA). Purified kinetochores were eluted from beads into 70 ml of 0.2% RapiGest (Waters Corporation) in 50 mM ammonium bicarbonate. This was achieved via gentle agitation on a Vortex-Genie 2 (Scientific Industries) set to the lowest setting for 30 minutes at room temperature. 60 ml of each elution sample was sent for mass spectrometry processing.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Saccharomyces Cerevisiae W303 (ncbitaxon:580240)

SUBMITTER: Susan Biggins

PROVIDER: MSV000097121 | MassIVE | Fri Feb 14 11:20:00 GMT 2025

SECONDARY ACCESSION(S): PXD060835

REPOSITORIES: MassIVE

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Analysis of a cancer-associated mutation in the budding yeast Nuf2 kinetochore protein

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