Figure 1. PDE12 is essential for efficient mitochondrial translation.

(A) Western blotting for steady-state levels of OxPhos components in PDE12+/+, PDE12 ± and PDE12−/− cells, and in PDE12−/− expressing either wild-type PDE12 or the E351A catalytic mutant at increasing expression levels. (B) Basal oxygen consumption of PDE12+/+ and PDE12−/− cells, and PDE12−/− cells expressing either wild-type PDE12 or E351A. Mean values ± SEM are shown, n = 3. *p<0.05, ***p<0.001 with Student t-test relative to PDE12+/+. (p values: [+/+] vs. [+/−] p=0.0141; [+/+] vs. [−/−] p=0.00134; [+/+] vs. [−/− + WT] p=0.12; [+/+] vs. [−/− + E351A] p=0.0144). (C) Growth curve of PDE12+/+, PDE12+/− and PDE12−/− cells, and PDE12−/− cells expressing either PDE12 wild-type or E351A cultured in DMEM containing 0.9 g/L galactose. Representative experiment is shown where each cell line was plated in quadruplicate (mean ± 1 SD). (D) Metabolic labelling of mitochondrial translation products with [35S]-methionine in PDE12+/+, PDE12+/− and PDE12−/− cells, and in PDE12−/− cells expressing either wild-type PDE12 or E351A for 24 hr at increasing levels. CBS: Coomassie blue stained gel as loading control. (E) Quantification of a subset of mitochondrial translation products in PDE12+/− and PDE12−/− relative to PDE12+/+using ImageQuant software. Relative quantification for each polypeptide was plotted against expected length of polypeptide. For COII/III, ND1/ND2 and COI/ND4 products were quantified together due to proximity of their bands on the gel, and plotted mass is the mean average of both proteins (mean ± SD; n = 6, for PDE12+/− n = 3, **p<0.01, ***p<0.001). (p values: [+/+] vs. [+/−]: ATP8 p=0.148, ND4L p=0.407, ND3 p=0.299, COX2/COX3 p=0.263, ND1/ND2 p=0.397, COX1/ND4 p=0.12124, ND5 p=0.08528; [+/+] vs. [+/+]: ATP8 p=0.328, ND4L p=0.058, ND3 p=0.00857, COX2/COX3 p=9.21×10⁻⁵, ND1/ND2 p=0.00029, COX1/ND4 p=2.08×10⁻⁵, ND5 p=0.00016.

DOI: http://dx.doi.org/10.7554/eLife.27596.002

Figure 1—figure supplement 1. Gene targeting of PDE12 with ZFNs and complementation.

(A) Schematic of PDE12 gene locus indicating target sites of ZFNs. The indels present in clones 71 (PDE12−/−) or 118 (PDE12+/−) are indicated. (B) Western to confirm knockout of PDE12 in biallelic PDE12−/− cell line (clone 71). (C) Western blotting of four additional PDE12−/− clones produced by re-transfection of PDE12+/− (clone 118) to confirm knockout and reduction in steady-state levels of OxPhos subunits. (D) Mitochondrial translation products were labelled with [³⁵S]-methionine in PDE12+/+ and four additional PDE12−/− clones. CBS- Coomassie blue stained gel as loading control. (E) Immunofluorescence to confirm exclusion of Δ16 and Δ23 PDE12 variants from mitochondria in human cells due to loss of mitochondrial targeting sequence. TOM20, translocator of the outer mitochondrial membrane 20. Scale bar = 20 μm. (F) Western to confirm PDE12−/− cells requires expression of catalytically active PDE12 to be targeted to mitochondria for recovery of steady-state levels of OxPhos subunits.

DOI: http://dx.doi.org/10.7554/eLife.27596.003