Dataset Information

Native Purification of yeast Met30 in the absence and presence of Cadmium followed by DSSO crosslinking and Mass spec analysis

ABSTRACT: HBTHMet30 cells were cultured in YEP + 2% galactose + 4µM Biotin at 30˚C and treated with 100 µM CdCl2 for 30 minutes. Native whole cells lysates were prepared in lysis buffer (50mM Hepes pH7.5, 200mM NaCl, 10% glycerol, 40mM imidazole, 0.2% Triton1mM dithiothreitol, 0.1mM orthovanadate, 1mM phenylmethylsulfonyl floride [PMSF], and 1mg/ml each leupeptin and pepstatin) and bound to Ni-resin. Proteins were eluted in the presence of 250mM imidazole. For cross-linking analysis, eluted HBTHMet30 was first bound to Streptavidin beads and then on-bead cross-linked with 0.5 mM DSSO in PBS buffer for 1 h at 37 °C. Bead-bound proteins were reduced with TCEP and alkylated with iodocetamide, digested by trypsin and cleaned-up with C18 tips prior to LC MSn analysis.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Clinton Yu

LAB HEAD: Peter Kaiser

PROVIDER: PXD048194 | Pride | 2024-06-22

REPOSITORIES: Pride

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Publications

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

Lauinger Linda L Andronicos Anna A Flick Karin K Yu Clinton C Durairaj Geetha G Huang Lan L Kaiser Peter P

Nature communications 20240508 1

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCF<sup>Met30</sup> ubiquitin ligase during cadmium stress. Several highly c ...[more]

PMID: 38719837

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Dataset Information

Native Purification of yeast Met30 in the absence and presence of Cadmium followed by DSSO crosslinking and Mass spec analysis

Publications

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

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