Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7−/− mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7−/− tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.


Statistics
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The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
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Data collection
Snapgene was used to visualize the mouse genome at the Pptc7 locus and design gRNA and primer sequences. AMT Capture Engine software was used to capture electron micrographs. QuantStudio Real Time PCR system software was used to capture qPCR data. NIS Elements software was used to capture confocal flourescence images. Image Studio sofware (LiCOR) was used to capture Western blot images.

Data analysis
Microsoft excel was used to analyze data, generate volcano plots, standard bar graphs, and to perform Student's t tests. ImageJ was used to quantify mitochondrial area in Figures 3H and K, and to quantify band intensities in Figures 5 and S5. BoxPlotR, a web-tool, was used to analyze data to generate boxplots in Figures 2A-E, 2H, 5P, S2G-I. Coon OMSSA Proteomic Analysis Software Suite (COMPASS) was used to search RAW files for proteomic and phosphorproteomic data.
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Data
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Sample size
Select biological measurements were taken for all mice studied (e.g. blood glucose, weight), and were included in the analysis. For other biological measurements (e.g. ketone measurements, lactate, acylcarnitine analysis, proteomics/phosphoproteomics), at least 5 biological replicates of wild type and Pptc7 knockout mice were used, which allowed a fold change of 0.5 to be calculated as significant (p<0.05; Type I error = 0.05; power 90%).
Data exclusions In the acylcarnitine data analysis, 4 datapoints (with a datapoint defined as a single measurement in one acylcarnitine species from one biological replicate) were dropped due to being extreme outliers; outliers were tested identified using Grubbs' test via Graphpad software.

Replication
For physiological measurements (e.g. serum ketones), acylcarnitine analysis, metabolomic analysis, proteomic and phosphoproteomic analysis, mice were genotyped and chosen across multiple, independent litters for each experiment. For the biological follow up experiments ( Figures 5 and 6), Tim50p mutant strain import assays ( Figure 5) and import assays with HADH ( Figure 6) were carried out by two independent laboratories (NMN in the Pagliarini lab and LM/FNV at the University of Frieberg) with similar reproducible results.
Randomization Mice were sac'd at birth and randomly assigned a number. Tail tips were collected, genotyped, and the at least 5 mice from each genotype were randomly selected from this pool for each experiment, allowing randomization across litters (and likely sexes, as the sex of perinatal pups is indistinguishable at birth).

Blinding
Due to the randomized numbering system described above, data analysis was done blinded and deconvoluted by genotype after data were collected.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation For FLAG: non-epitope expressing cell line used as negative control to ensure band specificity; various constructs used with different molecular weights, which were used for identification of the expected protein size.

October 2018
For VDAC, OxPhos, Timm50, actin, GRP75, PMPCB, Por1p, tubulin -bands identified at expected molecular weights for protein products. ; two strains are registered as two independently segregating Pptc7-/-alleles were generated in our founder mouse -one with a 4 bp deletion in exon 2 (hereby called E2; MGI:6094244), and one with a 1 bp deletion in exon 3 (hereby called E3; MGI:6143811). Wild type C57BL/6J mice were used for propagation of the strains as necessary.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not include field-collected samples.

Ethics oversight
All animal work complied with ethical regulations for animal testing and research, and was done in accordance with IACUC approval by the College of Agricultural and Life Sciences (CALS) Animal Care and Use Committee at the University of Wisconin-Madison (protocol/animal welfare assurance #A3368-01).
Note that full information on the approval of the study protocol must also be provided in the manuscript.