Supplementary Figure 2: Identifying a non-nuclear mutant of COQ7.
(a) Alignment of mammalian COQ7 N-terminal protein sequences (amino acids 1 to 42) using Clone Manager (Sci-Ed Software); HS, Homo sapiens; CL, Canis lupus familiaris; MM, Mus musculus; RN, Rattus norvegicus. Conserved residues are in red and R28 is in blue. MPP marks the predicted mitochondrial processing peptidase cleavage site. Residues mutated and assessed in the fluorescence studies in panel b are denoted with asterisks. (b) Point mutations in the COQ7 N-terminus cause reduced nuclear localisation. GFP fluorescence in COS7 cells expressing COQ7 fused at the C-terminus to GFP and harboring the point mutations R21A, Y26F and R28A were analysed. Quantification of the percent of cells displaying nuclear staining is shown (100 cells assessed in each of n = 3 independent experiments; error bars, s.e.m. ∗∗P < 0.005). The most severe loss of nuclear staining was observed with the R28A mutation. (c) COQ7 (R28A) mutant displays reduced levels of the uncleaved form. Lysates from HEK293 cells expressing OLLAS and FLAG tagged COQ7 (COQ7O/F) or COQ7(R28A)O/F (Fig. 2b) were immunoblotted with anti-COQ7 antibody. (d) Parent HEK293 cells (Ctrl) or cells stably expressing untagged (WT) or non-nuclear COQ7 (R28A) were transfected with siCTRL or siCOQ7that specifically targets endogenous COQ7 mRNA. Transcript levels of endogenous COQ7 mRNA (5′UTR amplicon) were analysed (mean values from 3 reactions per condition in n = 4 independent experiments; error bars, s.e.m. ∗∗P < 0.005). (e) Reverse phase HPLC chromatograms of quinones. Purified ubiquinone-10 (UQ10) was used as a standard. Levels of UQ10 and demethoxyubiquinone-10 (DMQ10) were measured in HEK293 cells treated with the COQ7 inhibitor clioquinol (CQ; 10 μM, 24 h), or from WT or R28A expressing HEK293 cells. CQ caused the appearance of DMQ10. UQ10 peak at 8.63 min, DMQ10 peak at 8.39 min. (f) Levels of lactate dehydrogenase (LDH) in media from cultured WT and R28A cells are similar, indicating that cell survival under basal conditions is not changed (mean values from 4 wells of cells per condition in n = 3 independent experiments; error bars, s.e.m. ∗∗P < 0.005). Treatment with 0.1% (v/v) Triton X-100 (TX) for 30 min was used as a positive control.
