Fig. 8: Import deficient COXIV is ubiquitinated and interacts with TbUbL1.

a Top panel, immunoblots of whole cell extracts of uninduced and induced atypical translocase of the outer membrane 69 (ATOM69) RNAi cell lines that simultaneously express c-terminally myc-tagged cytochrome oxidase subunit IV (COXIV-myc) or a derivative thereof that lacks the mitochondrial targeting sequence (ΔMTS-COXIV-myc). The immunoblot was probed for the myc-tagged COXIV variants, ATOM69 and elongation factor 1a (EF1a), which serves as a loading control. Bottom panel, quantifications of the levels of the myc-tagged COXIV variants from triplicate immunoblots shown in the top panel, with the COXIV-myc level of each uninduced cell line set to 100%. The level of COXIV-myc for each sample was normalised to its respective EF1a signal. Data are presented as mean values with error bars corresponding to the standard deviation of the mean from three independent biological replicates. Significance calculated using a two-tailed unpaired t-test, with the p values calculated indicated in the graph. b Immunoprecipitation (IP) using ubiquitin-binding Ubiqapture beads of whole cell extracts of the uninduced and induced (3d) ATOM69 RNAi cell line that expresses ΔMTS-COXIV-myc. 5% whole cell extracts (IN), 5% of the unbound protein fraction (UB) and 50% of the final eluate (IP) were separated by SDS PAGE. The resulting immunoblots were probed with an antiserum against ubiquitin (left panel, Ub) or for ΔMTS-COXIV-myc (right panel, myc). ATOM40 (bottom panel) is used as a negative control. Arrow indicates a band of monoubiquitinated ΔMTS-COXIV-myc detectable by both anti-Ub and anti-myc. This IP analysis was repeated independently twice with similar results. c IP using myc conjugated beads with whole cell extracts of the uninduced and induced (3d) ATOM69 RNAi cell line that expresses ΔMTS-COXIV-myc. 5% whole cell extracts (IN), 5% of the unbound protein fraction (UB) and 50% of the final eluate (IP) were separated by SDS-PAGE. The resulting immunoblots were probed for the myc-tagged COXIV variants (top panel), TbUbL1 (middle panel) and VDAC, which serves as a loading control (bottom panel). This IP analysis was repeated independently twice with similar results. Source data are provided as a Source Data file.