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Fig. 2 | BMC Biology

Fig. 2

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

Fig. 2

Disturbance of Cox12 protein oxidative folding triggers its rapid proteasome-mediated degradation. a Schematic illustration of the cycloheximide (CHX) chase experiments. b Degradation of Cox12FLAG with or without 5 mM DTT treatment, tested using CHX chase. DTT was added in parallel to CHX as indicated. c Schematic representation of Cox12 protein with indicated positions of cysteine residues and disulfide bonds. d Growth test of WT and Δcox12 yeast transformed with a plasmid that carried Cox12FLAG or Cox12C-FREE-FLAG under control of the galactose-inducible promoter or an empty vector control. Tenfold dilutions were spotted on selective minimal medium plates with a carbon source as indicated and grown at 28 °C. e Import of radiolabeled Cox12 or Cox12C-FREE into mitochondria isolated from WT cells. The incubation times are indicated. Pretreatment with 50 mM IA was used as a negative control. f Degradation of Cox12FLAG and Cox12C-FREE-FLAG, tested using CHX chase with or without DTT. g mCherry/sfGFP fluorescent signal ratio measured in WT cells that expressed plasmid-borne Cox12-tFT, Cox12C-FREE-tFT, and empty tFT. Cells were cultured in selective medium with glucose at 28 °C. The fluorescence ratio for the empty tFT was set to 1. Data are expressed as mean ± SEM, n = 6. Cox12-tFT and empty tFT data are the same as in Fig. 1d. h, i The degradation of Cox12FLAG (h) or Cox12C-FREE-FLAG (i) in WT or pre2-DAmP yeast, tested using CHX chase. In parallel to CHX, 5 mM DTT was added (h). b, f, h, i The plasmid-borne Cox12FLAG was expressed using the copper-inducible promoter; yeast were cultured in selective medium with 2% glucose at 28 °C. Proteins were analyzed by SDS-PAGE and immunodetection (b, f, h, i) or autoradiography (e). CHX, cycloheximide; DTT, dithiothreitol; ev, empty vector; IA, iodoacetamide; PK, proteinase K; WT, wild-type

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