Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy

Abstract

Pptc7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass concomitant with elevation of the mitophagy receptors Bnip3 and Nix. Consistently, Pptc7^-/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs-including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

Figure 1:. Acute knockout of Pptc7 compromises hepatic mitochondrial content in adult mice.

A. Schematic for the generation of a conditional Pptc7 mouse to allow global, inducible knockout in adult mice. B. Genotyping verification of Cre-mediated excision of Pptc7 exon 3 after tamoxifen treatment. C. Schematic for the 16plex proteomic analysis of liver tissue from control (UBC-ER^T2-Cre;Pptc7^+/+) or experimental (UBC-ER^T2-Cre;Pptc7^flox/flox) animals. D. Proteomic analysis of non-mitochondrial (black dots) and mitochondrial (purple dots) proteins across 16 liver samples. E. Linear regression of the fold changes in mitochondrial proteins identified in both adult inducible KO liver (y-axis) and perinatal KO liver (x-axis).

Figure 2:. Pptc7 knockout causes cell-autonomous decreases in mitochondrial protein content leading to broad metabolic defects.

A. Genotyping of wild type (WT) and Pptc7 KO mouse embryonic fibroblasts (MEFs). B. Pptc7 endogenous protein expression in WT and KO MEFs. Notably, Pptc7 runs as a set of two doublets, marked by arrows. A non-specific band (*) serves as a loading control. C. Proteomic analysis of non-mitochondrial (black dots) and mitochondrial (green dots) proteins from triplicate WT and Pptc7 KO MEFs. The mitophagy receptors Bnip3 and Nix are highlighted. D. Fold changes of mitochondrial proteins across all Pptc7 KO systems including perinatal heart (orange) and liver (blue), inducible adult liver (purple), and MEFs (green). In each system, loss of Pptc7 causes significant decreases in the mitochondrial proteome relative to non-mitochondrial proteins (shown in grey for all systems). ***p<0.001, one way ANOVA; multiple comparisons across each paired WT and KO dataset. Mean and standard deviation shown. E. Seahorse analysis of a mitochondrial stress test performed on primary Pptc7 KO MEFs grown in standard DMEM supplemented with 25 mM glucose. F., G. Seahorse analysis of a mitochondrial stress test performed on primary permeabilized Pptc7 KO MEFs given pyruvate and malate (F.) or succinate and rotenone (G). For all Seahorse analyses, error bars represent standard deviation. H.,I. BioLog analysis of permeabilized wild type and Pptc7 KO MEFs given various TCA cycle substrates (H.) or amino acids and fatty acids (I.). For BioLog analysis, each datapoint represents an independent well on the BioLog plate, with the mean shown. Error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.001; BioLog data was analyzed using a Student’s t test.

Figure 3:. Pptc7 knockout causes excessive Bnip3- and Nix-mediated mitophagy.

A. Western blot of Bnip3 and Nix expression in wild type, Pptc7 KO, and two Pptc7/Bnip3/Bnip3l triple knockout (TKO) MEF cell lines. Actin is shown as a load control. B. Representative images of wild type, Pptc7 KO, and each TKO cell line expressing the mitophagy reporter mt-Keima. Mitochondria imaged at 458 nm are at physiological pH but those imaged at 561 nm reflect acidic mitochondria undergoing mitophagy. C. Quantification of mt-Keima imaging. Each grey dot represents the ratio of mitochondrial fluorescence from a single cell. Blue, orange, and purple dots represent averages from three independent biological experiments. Error bars represent standard deviation. ****p<0.0001. mt-Keima data analyzed by one way ANOVA. D., E. Proteomic analysis of mitochondrial proteins in two independent Pptc7/Bnip3/Bnip3l TKO lines normalized to Pptc7 KO alone. F. Seahorse analysis of a mitochondrial stress test in immortalized wild type (WT, blue), Pptc7 KO (red), TKO #1 (dark blue), TKO #2 (teal). Cells were assayed in Seahorse DMEM supplemented with 25 mM glucose. Error bars represent standard deviation. G. Seahorse analysis of basal oxygen consumption rates (OCR) and extracellular acidification (ECAR). Error bars represent standard deviation. H., I. Bnip3 (H.) and Nix (I.) protein expression across 207 cell lines harboring monogenic mutations in genes encoding mitochondrial-localized proteins. Only Pptc7 KO increases both Bnip3 and Nix significantly across this dataset.

Figure 4:. Phosphoproteomic analysis of Pptc7 KO systems reveals candidate substrates, including Bnip3 and Nix.

A., B. Protein-normalized mitochondrial phosphoisoforms in Pptc7 KO MEFs (green, A.) and inducible adult liver tissue (purple, B.). C. Analysis of phosphoproteomes across systems reveals many unique (i.e., only identified in a single experimental system) phosphosites (p-sites), with four significantly upregulated phosphosites identified across all four experimental systems (shown in grey box). D. Analysis of overlapping phosphosites identified in perinatal liver tissue (y-axis) and inducible adult liver (x-axis) show strong correlation. E. Non-protein normalized mitochondrial phosphoisoforms show Bnip3 and Nix have significantly elevated phosphorylation events across all tested model systems.