Fig. 3
From: Determinants of the cytosolic turnover of mitochondrial intermembrane space proteins
Lysine residues are required for Cox12 ubiquitination and proteasomal degradation. a Experimental scheme of 6His-tagged ubiquitin affinity purification. b Affinity purification of ubiquitinated Cox12FLAG via 6His-ubiquitin. c Amino acid residue frequencies in Cox12 proteins among the selected fungal species. Amino acid numbering is according to S. cerevisiae protein. The data are presented in WebLogo format; cysteine and lysine residues are marked with red and blue, respectively. Positions of lysine residues that are present in S. cerevisiae Cox12 are indicated. d Growth test of WT and Δcox12 yeast transformed with a plasmid that carried Cox12FLAG or its mutant with all seven lysine residues mutated into arginine residues (Cox12K-FREE-FLAG) under the control of the galactose-inducible promoter and an empty vector control. Tenfold dilutions were spotted on selective minimal medium with a carbon source as indicated and grown at 28 °C. e Degradation of Cox12FLAG and Cox12K-FREE-FLAG, tested using CHX chase experiments. The plasmid-borne Cox12FLAG or Cox12K-FREE-FLAG were expressed in WT cells using the copper-inducible promoter. Yeast were cultured in selective medium with 2% glucose at 28 °C. Samples were collected at the indicated time points, starting from the addition of CHX and DTT. f Affinity purification of ubiquitinated Cox12FLAG and Cox12K-FREE-FLAG via 6His-tagged ubiquitin. g Affinity purification of ubiquitinated Cox12FLAG, Cox12K-FREE-FLAG, and Cox12K-FREE-FLAG variants with single lysine residues reintroduced at positions 25, 36, or 41. h Affinity purification of ubiquitinated Cox12FLAG or its variant that possessed only a single lysine residue at position 73. b, f, g, h The plasmid-borne Cox12FLAG variants were expressed in WT cells under control of the galactose-inducible promoter. Yeast were cultured in modified selective medium with 3% glycerol at 28 °C. b, e, f, g, h Proteins were analyzed by SDS-PAGE and immunodetection. 6His-Ub, 6His-tagged ubiquitin; CHX, cycloheximide; DTT, dithiothreitol; ev, empty vector; WT, wild-type