Fig. 7

Cox12 protein ubiquitination prevents its import into mitochondria. a Juxtaposition of the three-dimensional density map of ubiquitin [PDB:1UBQ structure] and cross-section of the three-dimensional density map of the Tom40 protein-conducting channel based on homology modeling. b Import of radiolabeled Cox12, Cox12 head-to-tail fusion with ubiquitin (Cox12-Ub), and ubiquitin (Ub) into the mitochondria isolated from WT cells. The incubation times are indicated. Pretreatment with 50 mM IA was used as a negative control for MIA-dependent import. c Cellular accumulation of Cox12_FLAG, Cox12_C27S-FLAG, Cox12_C37S-FLAG, Cox12_C48S-FLAG, and Cox12_C59S-FLAG proteins in WT yeast with or without MG132 proteasome inhibitor treatment. Yeast were cultured in liquid modified selective medium with 3% glycerol at 28 °C; plasmid-borne Cox12_FLAG variants were expressed under the control of the galactose-inducible promoter. d Cellular accumulation of Cox12_C27S-FLAG or Cox12_C37S-FLAG proteins in WT and Δubc4 yeast. Yeast were cultured in liquid selective medium with 3% glycerol at 28 °C; plasmid-borne Cox12_FLAG variants were expressed under the control of the galactose-inducible promoter. e Mitochondrial import of precursors of MIA substrate proteins is sensitive to ubiquitination. Ubiquitinated precursor proteins are rerouted from the mitochondrial import pathway to proteasomal degradation. Proteins were analyzed by SDS-PAGE and autoradiography (b) or immunodetection (c, d). IA, iodoacetamide; PK, proteinase K; Ub, ubiquitin; WT, wild-type