Fig. 7: Nuclear export of TbUbL1 is essential for proteasomal digestion of FtsH.

a Left, immunofluorescence (IF) analysis of a cell line allowing tetracycline-inducible expression of ubiquitin-like protein 1 (TbUbL1) containing a nuclear localisation signal (NLS) in front of the C-terminal myc-tag (TbUbL1-NLS-myc +Tet). Right panels, IF analysis of a cell line allowing tetracycline-inducible expression of TbUbL1 containing a NLS in front of the C-terminal myc-tag together with simultaneous RNAi of atypical translocase of the outer membrane 69 (ATOM69) (TbUbL1-NLS-myc/ATOM69-RNAi +Tet). Cells were induced for 3d. Scale bar 10 μm. The % of the population displaying a TbUb1-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 Immunoblots depicting the change in abundance of in situ tagged FtsH-HA and endogenous cytochrome oxidase subunit IV (COXIV) in whole cells after tetracycline-inducible RNAi-mediated ablation of ATOM69 alone (left panels), in combination with RNAi-mediated ablation of TbUbL1 (middle panel), or in combination with tetracycline-inducible expression of TbUbL1-NLS-myc (right panel) are shown. Note that for the first two cell lines, sections of blots already shown in Fig. 2a, b were used, with the anti-COXIV stained panels shown now in addition. 2 × 10⁶ cells were loaded per lane. Elongation factor 1a (EF1a) is used as a loading control. c Quantification of triplicate immunoblots shown in b, with the tagged protein level of each uninduced cell line set to 100%. The level of tagged protein 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, except for the ATOM69/TbUbL1-RNAi cell line where six replicates were used to assess FtsH-HA level, as also used in Fig. 3b. 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. d Immunoprecipitation using myc-conjugated beads with whole cell extracts of the TbUbL1-NLS-myc expressing cell line induced for 1d. 5% whole cell equivalent (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 anti-myc-tag antibodies (top panel), antisera against TbUbL1 (middle panel), and EF1a (bottom panel) which is used as a negative control. This IP analysis was repeated independently twice with similar results. Source data are provided as a Source Data file.