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Fig. 5 | BMC Biology

Fig. 5

From: Determinants of the cytosolic turnover of mitochondrial intermembrane space proteins

Fig. 5

Involvement of the E2 ubiquitin-conjugating enzyme Ubc4 and the E3 ubiquitin ligase Rsp5 in Cox12 ubiquitination and degradation. a, b Affinity purification of ubiquitinated Cox12FLAG via 6His-Ub from WT or Δubc4 yeast cells. The plasmid-borne Cox12FLAG was expressed under the control of a galactose-inducible promoter (a) or a copper-inducible promoter (b). Yeast were cultured at 28 °C in modified selective medium with 3% glycerol (a) or 2% glucose (b). Monoubiquitinated Cox12FLAG levels were quantified and normalized to ubiquitin-free Cox12FLAG levels from the load fractions (a). The level of normalized monoubiquitinated Cox12FLAG from WT yeast was set to 100%. Data are expressed as mean ± SEM. n = 3. c, d Degradation of Cox12FLAG (c) or Cox12C-FREE-FLAG (d) in WT and Δubc4 yeast, tested using CHX chase. The plasmid-borne Cox12FLAG and Cox12C-FREE-FLAG were expressed using the copper-inducible promoter. Yeast were cultured in selective medium with 2% glucose at 28 °C. e Cellular protein levels in WT and Δubc4 yeast grown in minimal medium with 3% glycerol at indicated temperatures. f Affinity purification of ubiquitinated Cox12FLAG via 6His-Ub in WT and rsp5-19 yeast. The plasmid-borne Cox12FLAG was expressed under the control of the copper-inducible promoter. Yeast were cultured in modified selective medium with 2% glucose at 37 °C. Degradation of Cox12FLAG (g) or Cox12C-FREE-FLAG (h) in WT and rsp5-19 yeast. The plasmid-borne Cox12FLAG or Cox12C-FREE-FLAG were expressed under the control of the copper-inducible promoter. Yeast were cultured in selective medium with 2% glucose at 28 °C. c, d, g, h Samples were collected at the indicated time points, starting from the addition of CHX. In c, g, 5 mM DTT was added in parallel to CHX. i Cellular protein levels in WT and rsp5-19 yeast grown in minimal medium with 3% glycerol at indicated temperatures. Proteins were analyzed by SDS-PAGE and immunodetection (all panels). 6His-Ub, 6His-tagged ubiquitin; CHX, cycloheximide; DTT, dithiothreitol; WT, wild-type

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