Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis

Abstract

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.

Figure 1. Inactivation of the Ptpmt1 gene leads to mouse embryonic lethality

A) Gene targeting strategy. Exons (blue boxes), translation initiation site (ATG), selection gene (neomycin, neo), positions of 5′ and 3′ probes for Southern blot (red bars), PCR primer (red arrows), loxP sites ( ), Frt sites ( ), and restriction sites are indicated. X-XbaI, E-EcoRI. B) Genotype of offspring from Ptpmt1 heterozygous intercrosses at various developmental stages. C) Derivation of Ptpmt1-deficient mouse embryonic fibroblasts. Ptpmt1^flox/flox MEFs were infected with recombinant adenoviruses encoding GFP(−) or GFP-Cre (+). Cells were harvested at the indicated time points post-infection for western blot analysis. dpi, days post infection. See also Figure S1.

Figure 2. Inhibition of mitochondrial respiration in Ptpmt1-deficient cells

A) Oxygen consumption rates (OCR) of intact MEFs cultured in regular medium (25mM glucose) at 13dpi. The basal rate, the State 4 rate (with 1uM oligomycin, Oligo), and the maximal uncoupler stimulated rate (in the presence of FCCP) are shown. Rotenone (Rot) and antimycin A (Anti A) were used to block all electron flow through Complex I and III, demonstrating that non-mitochondrial oxygen consumption in these cells is negligible. Mean ± SEM from four replicates. B) Whole cell ATP contents in MEFs (13dpi) cultured in regular medium or pre-incubated with 3mM glucose containing medium overnight. Mean ± S.D., n=3, **p<0.01. Maximal uncoupler stimulated respiration rates (C) and extracellular acidification rate (ECAR) rates (D) of Ptpmt1-Flox and KO MEFs preincubated with low glucose (3mM) media overnight are obtained at the indicated time post adenoviral infection. Mean ± SEM from four replicates. **p<0.01, *p<0.05. E) At 13dpi, crude mitochondria were isolated from Flox or KO MEFs, solubilized in β-dodecylmaltoside, subjected to BN-PAGE, and followed by consecutive Complex I and Complex II in-gel activity assays. The activities of each complex are indicated by the appearance of a pink colored band. See also Figure S2.

Figure 3. PTPMT1 maintains mitochondrial membrane integrity

A) Ptpmt1-deficient MEFs have fragmented mitochondria. Two weeks after deletion of Ptpmt1, cells were stained with anti-cytochrome c antibody and visualized by immunofluorescence microscopy. DNA was stained blue with 4′,6-diamidino-2-phenylindole (DAPI). B) Electron micrographs of Flox MEFs. Normal mitochondria with lamellar cristae are enlarged in the inset. Scale bar, 1μm. C) EM images of defective mitochondria in KO cells. Inset shows a vesicular mitochondrion and rupture of the outer mitochondrial membrane/OMM (red arrow). Red asterisk, loss of cristae and matrix components. D) Morphometric analysis of mitochondrial length. Mean ± SEM, n=80. E) Morphometric analysis of cristae/OMM surface area ratio. Mean ± SEM, n=52. ***p<0.001, **p<0.01.

Figure 4. Loss of PTPMT1 leads to the accumulation of phosphatidylglycerophosphate in vivo

Mass spectra of PGP in Flox (panel A) and KO (panel B) MEFs 13 days after deletion of Ptpmt1. Insets of panel A) and B) identified [M-2H]²⁻ ions of 34:1 PGP (exact mass m/z 413.239) at m/z 413.25, its ¹³C₁-isotope at m/z 413.75, ¹³C₂-isotope at m/z 414.25, and 34:2 PGP at m/z 412.24. The total number of carbons and unsaturations in the fatty acyl chains are listed to indicate the molecular species of each ion. See also Figure S3. C) and D) Mass spectra [M-H]⁻ ions of PG and [M-2H]²⁻ ions cardiolipin (CL) in Flox and KO MEFs, respectively. The scales of panel C and D are set at different levels to facilitate the observation of PG and cardiolipin ions in KO cells. E) Ratio of 34:1 PGP to 34:1 PG at the indicated time after Ptpmt1 deletion. F) Schematic diagram of cardiolipin biosynthesis pathway. PA, phosphatidic acid; CDS, CDP-DAG synthase; PGS, PGP synthase; CLS, cardiolipin synthase; TAZ, cardiolipin remodeling enzyme, Tafazzin. Enzymatic reactions that take place in mitochondrion are enclosed within the blue rectangle.

Figure 5. PTPMT1 dephosphorylates phosphatidylglycerophosphate in vitro

A) Conversion of ¹⁴C-labeled PGP to ¹⁴C-PG by recombinant wild type (WT) versus catalytically inactive (C132S) PTPMT1. A trace amount of radiolabeled lysoPG was generated during the reaction. B) The specific activities of PTEN, Laforin, DSP21, and VHR are compared with that of PTPMT1 using pNPP as the substrate. Mean ± S.D., n=3. C) PGP phosphatase activity is not a general feature of protein tyrosine phosphatases. The activity of each enzyme using PGP as the substrate is indicated by the absorbance at 620nm using malachite green reagents to measure the release of inorganic phosphate (Mean ± S.D., n=3).

Figure 6. Wildtype but not an active site mutant PTPMT1 rescues cardiolipin deficiency

A) The expression of β-galactosidase (LacZ), wildtype PTPMT1 (MT1), or catalytically inactive PTPMT1 mutant (CS). B) After labeling MEFs with ³²P-orthophosphate, phospholipids were extracted and the percentage of cardiolipin (CL) among total phospholipids was determined by TLC analysis. Mean ± S.E.M. from three independent experiments, one-way ANOVA with a post hoc F test, ***p<0.001, **p<0.01. C) The accumulation of PGP in Ptpmt1-deficient cells. For each sample, 50,000cpm of ³²P-labeled phospholipids were applied onto TLC plates and visualized by phosphoimager. D) Oxygen consumption rates of intact MEFs cultured in regular medium. The basal rate, the State 4 rate (Oligo), and the maximal uncoupler stimulated rate (FCCP) are shown as described in Figure 2. Mean ± SEM from four replicates. E) Wildtype but not mutant PTPMT1 complements the growth deficiency of yeast PGP phosphatase GEP4-null (gep4Δ) cells in a serial dilution spotting assay. The expression levels of FLAG-tagged GEP4 and PTPMT1 are indicated by western blot analysis. The level of CDC2 is shown as control. WT, wildtype strain. (−) empty vector. SCD, synthetic complete medium with dextrose. EtBr, Ethidium Bromide. See also Figure S4 and Table S1.