Fig. 4: Import defect induced by ATOM69 RNAi but not by other ATOM subunits causes a release of nuclear TbUbL1 to the cytosol.

a Left, immunofluorescence (IF) analysis of a cell line allowing tetracycline-inducible expression of myc-tagged ubiquitin-like protein 1 (TbUbL1-myc +Tet). Right, IF analysis of a cell line allowing tetracycline-inducible expression of myc-tagged TbUbL1 together with simultaneous RNAi of the atypical translocase of the outer membrane 69 (ATOM69) (TbUbL1-myc/ATOM69-RNAi +Tet). Scale bar 10 μm. Cells were induced for 3d. The % of the population displaying a TbUbL1-myc nuclear release phenotype is recorded below the images, 100 cells of each were analysed. This IF analysis was repeated independently at least three times with similar results. b IF analysis of cells expressing TbUbL1-myc with simultaneous RNAi of ATOM46, ATOM12 or ATOM69. Scale bar 10 μm. Cells were induced for 2d (cell lines based on ATOM12 RNAi) or 3d (cell lines based on ATOM46 or ATOM69 RNAi). The ATOM69 RNAi cell lines were combined with tetracycline-inducible expression of either N-terminally truncated (ΔN103-) or full length (FL-) ATOM69, respectively. This IF analysis was repeated independently at least two times with similar results. c Immunoblots depicting the change in abundance of the model substrate FtsH-HA in whole cells after tetracycline-inducible RNAi-mediated ablation of ATOM69, ATOM46 or ATOM12, or of ATOM69 in combination with tetracycline-inducible expression of either N-terminally truncated (ΔN103-) or full length (FL-) ATOM69 as indicated. Note that for the ATOM69 RNAi cell line, the sections of blots used are already shown in Fig. 2a. 2 × 10⁶ cells were loaded per lane. Elongation factor 1a (EF1a) is used as a loading control. d Quantification of triplicate immunoblots shown in c, with the FtsH-HA level of each uninduced cell line set to 100%. The level of FtsH-HA 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 of three independent biological replicates. Significance was calculated using a one way ANOVA followed by a Bonferroni posthoc test to allow for multiple comparisons, with the p values calculated indicated in the graph. Source data are provided as a Source Data file.