The UPR ^mt promotes mitochondrial import in vivo. (a and b) Subcellular fractionation and Western blot of mitochondrial chaperone HSP-6. glp-1 animals were grown at 25°C on bacteria expressing cco-1 dsRNA from hatching until the first day of adulthood. Control worms were grown on bacteria containing the empty RNAi vector alone. Different fractions were separated via differential centrifugation. (a) Representative blot of HSP-6, α-tubulin, and Ndufs-3. (b) Signal intensity was normalized to total protein level (Ponceau S staining). HSP-6 level in each fraction was then normalized to that of the total fraction in the empty RNAi vector control. The graph is presented as mean ± SEM, n = 3. Ratio paired t test (two-tailed) was performed for each subcellular fraction to compare the difference between HSP-6 with and without the induction of UPR^mt. *, P < 0.05; **, P < 0.01. (c and d) Immunostaining and confocal imaging of HSP-6p::HSP-6::GFP. (c) Representative images. Green, GFP; purple, ATP-5a. Scale bar = 5 μm. (d) Images were quantified with Fiji for total GFP and mitochondrial GFP (GFP within the boundary of mitochondrial marker ATP5a). The relative quantity of mitochondrial HSP-6::GFP was calculated as the ratio between mitochondrial and total GFP signals. This ratio for worms with different RNAi treatment was then normalized to the empty vector control. Mitochondrial HSP-6::GFP increased by 60% upon the induction of UPR^mt by cco-1 RNAi. This increase was partially suppressed by compromising mitochondrial import capacity through knocking down tomm-20. The graph is presented as mean ± SEM of three biological repeats. For each condition, total number of worms imaged was n ≥ 8, total number of images quantified n ≥ 15. t test (two-tailed) was performed to compare the difference between different treatments. *, P < 0.05; **, P < 0.01. (e and f) Comparison of fumarase activity in different subcellular fractions. (e) Fumarase activity in each fraction was compared for animal with or without the induction UPR^mt. Fumarase activity was normalized to the quantity of protein used in the assay. The graph is presented as mean ± SEM, n = 2. Paired t test (two-tailed) was performed for each subcellular fraction to compare the differences, **, P < 0.01. (f) Fumarase activity in each fraction was compared between cco-1 knocked down animals treated with or without tomm-20 RNAi. Histogram shows the changes of fumarase activity in each fraction, as represented by the ratio between animals treated with tomm-20 cco-1 double RNAi and cco-1 single RNAi (x–y intersection is set to [0,1] to better show the change in ratio). A mild yet consistent increase in cytosolic fraction and a corresponding decrease in the mitochondrial fraction were observed. The graph is presented as mean ± SEM, n = 3. Paired t test (two-tailed) was performed compare the difference between mitochondrial and cytosolic fractions. *, P < 0.05. Source data are available for this figure: SourceData F3.