Leader peptide or pro-segment mutants of renin are misrouted to mitochondria in autosomal dominant tubulointerstitial kidney disease

Figures

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  Fig. 1.. Prediction of the subcellular localisation of the indicated renin isoforms. Prediction was performed using the software WoLF PSORT. Uromodulin and paraplegin are shown as examples of a secreted and mitochondrial protein, respectively. Renin mutant isoforms p.L12P, p.L13Q, p.L16P, p.L16R, p.W17R and p.C20R have the highest score for mitochondrial localisation, whereas WT renin is predicted to be secreted. No clear effect is observed for mutations in the leader peptide W10R and L16del or in the pro-segment M39K and E48K, for which the prediction is similar to WT renin.

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  Fig. 2.. Mitochondrial targeting of mutant renin isoforms. (A) Western blot analysis showing renin expression in the lysates and conditioned media of transiently transfected Calu-6 cells. GAPDH is shown as a loading control. The presence of a non-glycosylated renin isoform (red asterisk) can be seen for all mutants. (B) Cell lysates were deglycosylated with PNGase F. The lower band indicated in panel A with the asterisk is not sensitive to PNGase F treatment, indicating the absence of N-glycans. (C) Immunofluorescence analysis in Calu-6 cells showing merged pictures of renin (red) and TIM44 (marker of mitochondria, green) with nuclei (DAPI, blue). Scale bar: 20 μm. Colocalisation of renin and mitochondria can be assessed by the presence of merged yellow signal. The graph below shows Pearson's correlation coefficient between signals of TIM44 and renin. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA with Bonferroni post hoc test versus WT. Data are shown as mean±s.e.m. (n=3 independent experiments). (D) Protease protection assay performed on isolated mitochondria from HEK293 cells expressing p.L16R or p.W17R mutant isoforms. Western blot experiments showed digestion of renin…

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  Fig. 3.. Identification of the domain necessary for mistargeting to mitochondria of mutated renin isoforms. Immunofluorescence analysis in Calu-6 cells showing merged pictures of the indicated GFP fusion constructs (green) and TIM44 (marker of mitochondria, red). Scale bar: 20 μm. A schematic representation of each construct is indicated above the picture. Leader peptide is depicted in blue, the pro-segment in pink and GFP in green. The red star indicates the insertion of an ADTKD mutation. Images are representative of three independent experiments.

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  Fig. 4.. Defective mitochondrial import upon expression of mutant renin isoforms. (A) Representative western blot analysis showing Mito-YFP when co-expressed with the indicated renin isoforms. ‘Val’ indicates cells treated with valinomycin as a positive control of defective mitochondrial import. (B) Quantification of Mito-YFP import, measured as the ratio between precursor (YFP with the MTS, upper band) and mature (YFP without the MTS, lower band) forms. **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA with Bonferroni post hoc test versus WT. Data are shown as mean±s.d. (n=4-14 independent experiments per group). (C) Immunofluorescence analysis showing merged images of the indicated renin construct (green), a mitochondrial marker (TIM44, in red) and nuclei (DAPI, blue). Yellow signals indicate the colocalisation of renin with mitochondria. Scale bar: 20 μm. A schematic of the transfected constructs is shown above each image. The leader peptide is depicted in blue, the pro-segment in pink, mature renin in purple and the MTS in red. The red star indicates the insertion of p.L16R mutation. MTS-REN is localised in the mitochondria as the p.L16R renin isoform. (D) Representative western blot…

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  Fig. 5.. Fragmentation of the mitochondrial network upon expression of renin mutants in the leader peptide or pro-segment. (A) Analysis of the morphology of the mitochondrial network in Calu-6 cells expressing the indicated renin isoforms. Cells were divided in three classes based on the pattern of expression of YFP targeted to mitochondria when co-transfected with the indicated renin isoforms. Representative images of YFP in cells corresponding to each class are shown. Scale bar: 10 μm. **P<0.01; ***P<0.001; ****P<0.0001; two-way ANOVA with Bonferroni post hoc test (versus WT fragmented). Data are shown as mean±s.e.m. (n=3-15 independent experiments per group). (B) Morphology of the mitochondrial network was determined by analysing the pattern of expression of the YFP targeted to mitochondria when co-transfected with the indicated constructs. The mature part of renin is not necessary for mitochondrial fragmentation. **P<0.01; ****P<0.0001; two-way ANOVA with Bonferroni post hoc test (versus WT fragmented). Data are shown as mean±s.e.m. (n=3-9 independent experiments per group). (C) Morphology of the mitochondrial network was determined by analysing the pattern of expression of the YFP…

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  Fig. 6.. Mitochondrial mistargeting of WT renin upon inhibition of ER translocation. (A) Immunofluorescence showing merged images of the indicated renin constructs (green), a mitochondrial marker (TIM44, in red) and nuclei (DAPI, blue). Yellow colour indicates colocalisation of renin or GFP fused proteins with mitochondria. Scale bar: 20 μm. (B) Morphology of the mitochondrial network was determined by analysing the pattern of expression of YFP targeted to mitochondria when co-transfected with the indicated renin isoforms. ****P<0.0001; two-way ANOVA with Bonferroni post hoc test (versus WT fragmented). Data are shown as mean±s.e.m. (n=2-6 independent experiments per group).

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  Fig. 7.. Renin mutants do not have a dominant negative effect on trafficking and secretion of WT renin. (A) Western blot analysis of co-expressed Flag- and HA-tagged renin isoforms in lysates and conditioned media of transfected CALU6 cells. GFP is shown as a control of transfection efficiency. (B) Immunofluorescence analysis showing the merged picture of Flag (green) and HA (red) renin isoforms in CALU6 cells. Wild-type renin cellular distribution is not affected by mutant renin co-expression. Scale bar: 15 µm. Images are representative of three independent experiments.

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  Fig. 8.. Summary of the effects of preprorenin mutations depending on their position in the protein domains. ‘+’ and ‘−’ indicate the presence and absence of the phenotype, respectively. NA, not assessed. A schematic representation of each construct is shown. The leader peptide is depicted in blue, the pro-segment in pink, a canonical mitochondrial targeting sequence (MTS) in red, the mature part of renin in purple and GFP in green. The red star indicates the insertion of an ADTKD mutation.