Fig. 6

Less-efficient import to mitochondria results in increased protein ubiquitination and decreased cellular accumulation. a Cellular accumulation of Cox12_FLAG, Cox12_C27S-FLAG, Cox12_C37S-FLAG, Cox12_C48S-FLAG, Cox12_C59S-FLAG, and Cox12_C-FREE-FLAG proteins in WT yeast. Yeast were cultured on selective minimal medium supplemented with glycerol as a carbon source at 28 °C. The expression of plasmid-borne Cox12_FLAG variants was driven by the galactose-inducible promoter. To induce expression, the medium was supplemented with 0.5% galactose for 6 h. b Growth test of WT and Δcox12 yeast transformed with plasmids that carried Cox12_FLAG or one of its single cysteine residue mutants (C27S, C37S, C48S, C59S) and an empty vector control. Tenfold dilutions were spotted on selective minimal medium with a carbon source as indicated and grown at 28 °C. c Import of radiolabeled Cox12, Cox12_C27S-FLAG, or Cox12_C37S-FLAG into mitochondria isolated from WT cells. Incubation times are indicated. Pretreatment with 50 mM IA was used as a negative control. The results were quantified, and the amount of WT Cox12 that was imported during 27 min of incubation was set to 100%. The data are expressed as mean ± SEM, n = 3. d, e Affinity purification of ubiquitinated Cox12_FLAG and Cox12_C27S-FLAG, Cox12_C37S-FLAG (d), or Cox12_C-FREE-FLAG (e) from WT cells via 6His-Ub. Levels of monoubiquitinated Cox12_FLAG variants were quantified and normalized to ubiquitin-free Cox12_FLAG variants from the load fractions. The ubiquitinated Cox12_FLAG level was set to 100%. The data are expressed as mean ± SEM. n = 3. Yeast were cultured in liquid modified selective medium with 3% glycerol at 28 °C, and plasmid-borne Cox12_FLAG variants were expressed under the control of the galactose-inducible promoter. Proteins were analyzed by SDS-PAGE and immunodetection (a, d, e) or autoradiography (c). 6His-Ub, 6His-tagged ubiquitin; ev, empty vector; IA, iodoacetamide; PK, proteinase K; WT, wild-type