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Mitochondrial Phosphatase PTPMT1 Is Essential for Cardiolipin Biosynthesis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell Metabolism Article Mitochondrial Phosphatase PTPMT1 Is Essential for Cardiolipin Biosynthesis Ji Zhang,1 Ziqiang Guan,5 Anne N. Murphy,1 Sandra E. Wiley,1 Guy A. Perkins,6 Carolyn A. Worby,1 James L. Engel,1 Philip Heacock,7 Oanh Kim Nguyen,1 Jonathan H. Wang,1 Christian R.H. Raetz,5 William Dowhan,7 and Jack E. Dixon1,2,3,4,* 1Department of Pharmacology 2Department of Cellular and Molecular Medicine 3Department of Chemistry and Biochemistry University of California, San Diego, La Jolla, CA 92093, USA 4Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA 5Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA 6National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093-0608, USA 7Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX 77030, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2011.04.007 SUMMARY importance of PTEN in tumor biology prompted us to identify additional phosphatases similar to PTEN (Pagliarini et al., PTPMT1 was the first protein tyrosine phosphatase 2004). We used the amino acid sequence surrounding the active found localized to the mitochondria, but its biological site, HCKAGKGR, which includes two invariant basic amino function was unknown. Herein, we demonstrate acids (bold) along with the catalytically essential Cys-X5-Arg that whole body deletion of Ptpmt1 in mice leads to (CX5R) motif, to search for closely related phosphatases. To embryonic lethality, suggesting an indispensable our surprise, we discovered a protein tyrosine phosphatase role for PTPMT1 during development. Ptpmt1 defi- (PTP) that localized exclusively to the inner mitochondrial membrane (Pagliarini et al., 2005). This was the first CX R ciency in mouse embryonic fibroblasts compromises 5 phosphatase found in mitochondria, and, because of its unique mitochondrial respiration and results in abnormal localization, we named it PTPMT1 (PTP localized to the Ptpmt1 mitochondrial morphology. Lipid analysis of - mitochondrion 1). This phosphatase is highly conserved and is deficient fibroblasts reveals an accumulation of present in animals, plants, and bacteria (Pagliarini et al., 2004). phosphatidylglycerophosphate (PGP) along with Mitochondria are ubiquitous and dynamic organelles that a concomitant decrease in phosphatidylglycerol. participate in crucial cellular processes in eukaryotic organisms. PGP is an essential intermediate in the biosynthetic They are responsible for the production of over 90% of cellular pathway of cardiolipin, a mitochondrial-specific ATP (Voet and Voet, 2004). Mitochondria are also the sites of phospholipid regulating the membrane integrity and fatty acid oxidation, ketone body production, heme biosyn- activities of the organelle. We further demonstrate thesis, cardiolipin metabolism, and coenzyme Q synthesis. In that PTPMT1 specifically dephosphorylates PGP addition, mitochondria are a major source of reactive oxygen species. They harbor key steps of gluconeogenesis and the in vitro. Loss of PTPMT1 leads to dramatic diminu- urea cycle and are central to the mechanisms of apoptosis (New- tion of cardiolipin, which can be partially reversed meyer and Ferguson-Miller, 2003; Voet and Voet, 2004). by the expression of catalytic active PTPMT1. Our Due to the unique localization of PTPMT1 in the inner mito- study identifies PTPMT1 as the mammalian PGP chondrial membrane, we postulated that it plays important roles phosphatase and points to its role as a regulator of in oxidative phosphorylation or other mitochondrial functions. cardiolipin biosynthesis. Therefore, we sought to elucidate the cellular function(s) and identify the endogenous substrate(s) of this phosphatase. INTRODUCTION RESULTS Reversible phosphorylation is a major regulatory mechanism controlled by more than 500 kinases and 100 phosphatases Inactivation of the Ptpmt1 Gene Leads to Embryonic (Alonso et al., 2004; Manning et al., 2002). Although the majority Lethality of kinases and phosphatases function on protein substrates, To study the physiological function(s) of PTPMT1, we genetically there are notable examples of kinases and phosphatases that engineered mice in which the Ptpmt1 gene was ablated through regulate the production and breakdown of phospholipids. For homologous recombination (Figure 1A). Subsequently, we inter- instance, phosphoinositide 3-kinase (PI3K) and PTEN are two crossed Ptpmt1-heterozygous mice to examine the conse- enzymes that regulate the abundance of key cellular phospho- quences of PTPMT1 deficiency. Out of 173 offspring born, lipids (Maehama and Dixon, 1998; Whitman et al., 1988). The 66 were wild-type, 107 were heterozygous, and none were 690 Cell Metabolism 13, 690–700, June 8, 2011 ª2011 Elsevier Inc. Cell Metabolism PTPMT1 Is the Mammalian PGP Phosphatase Inhibition of Mitochondrial Respiration in Cells Lacking PTPMT1 To further investigate the physiological functions of PTPMT1, we generated a floxed allele of Ptpmt1 in mice for Cre-mediated conditional knockout (Ptpmt1+/flox)(Figure 1A and Figure S1B). We next derived Ptpmt1flox/flox mouse embryonic fibroblasts (MEFs) from E13.5 embryos obtained from intercrosses of Ptpmt1+/flox mice. The cells were then infected with control adenovirus or adenovirus expressing Cre recombinase. The expression of PTPMT1 protein was almost completely ablated in the Cre-treated (KO) cells 3 days after the infection and remained negligible for at least 2 weeks in culture (Figure 1C). The level of PTPMT1 mRNA transcripts was also greatly dimin- ished in those cells (Figure S1C). MEFs lacking PTPMT1 grow slowly. Three days after infection with Cre recombinase, the proliferation rate of KO cells was reduced 25% compared to that of control adenovirus-treated cells (Flox) (Figure S2A). Inhibition of cell growth was more pronounced in KO MEFs at day 6. A colony formation assay was also performed to examine the long-term effect of PTPMT1 deficiency on cell growth. Not surprisingly, KO MEFs formed fewer and smaller colonies than Flox cells (Figures S2B and S2C). It is noteworthy that Ptpmt1 null cells were not apoptotic or necrotic (data not shown) and had cell sizes similar to Flox MEFs (Figure S2D). The position of PTPMT1 on the inner mitochondrial membrane places it within close proximity to electron transport chain complexes and enzymes of the tricarboxylic acid cycle. Therefore, we investigated the effects of PTPMT1 deletion on mitochondrial respiration and oxidative phosphorylation. Intact cell oxygen consumption rates (OCR) were measured at 13 days after deletion of Ptpmt1 (Figure 2A). The basal respira- tion rates were substantially lower in Ptpmt1 KO cells relative to that of Flox MEFs. We also assessed state 4 rates (a measure- Figure 1. Inactivation of the Ptpmt1 Gene Leads to Mouse Embry- ment of proton leakage across the inner membrane) by the onic Lethality addition of the ATP synthase inhibitor oligomycin, and maximal (A) Gene targeting strategy. Exons (blue boxes), translation initiation site (ATG), respiratory rates by the addition of the uncoupler, carbonylcya- selection gene (neomycin, neo), positions of 50 and 30 probes for Southern blot nide-r-trifluoromethoxyphenylhydrazone (FCCP). A dramatic (red bars), PCR primer (red arrows), loxP sites (blue triangles), Frt sites (green reduction in maximal respiratory rates as well as state 4 rates triangles), and restriction sites are indicated. X-XbaI, E-EcoRI. was observed in the KO cells (Figure 2A), suggesting that (B) Genotype of offspring from Ptpmt1 heterozygous intercrosses at various PTPMT1 is required for optimal mitochondrial respiration. developmental stages. (C) Derivation of Ptpmt1-deficient mouse embryonic fibroblasts. Ptpmt1flox/flox In contrast, when we concomitantly measured the cellular MEFs were infected with recombinant adenoviruses encoding GFP(–) or ATP content, an increase in ATP level was observed in GFP-Cre (+). Cells were harvested at the indicated time points postinfection for Ptpmt1-deficient MEFs. The increase was more evident after western blot analysis. dpi, days postinfection. preincubation of cells with low glucose medium (Figure 2B), See also Figure S1. consistent with our previous report that suppression of PTPMT1 expression by small-interfering RNA (siRNA) markedly homozygous null (Figure 1B) (see Figure S1 available online for enhances ATP levels in INS-1 832/13 cells in response to low detailed characterization of mouse genotyping), indicating glucose exposure (Pagliarini et al., 2005). Mitochondrial oxida- a Mendelian ratio typical of embryonic lethality. We next con- tive phosphorylation is a major source of cellular ATP produc- ducted timed mating experiments to determine the stage of tion; however, it is known that cells can modulate the balance the embryonic lethality. Embryos from E8.5, E10.5, E14.5, and between aerobic and anaerobic (glycolytic) ATP production E17.5–18.5 gestational stages were primarily wild-type or (Warburg, 1956) and that lowering extracellular glucose heterozygous for the Ptpmt1 allele (Figure 1B). Further dissection enhances glucose transport and utilization (Kletzien and identified an average of 26% resorption sites on the uterus Perdue, 1975). The diminished mitochondrial respiratory lacking discernible embryos from all developmental stages capacity in the presence of an elevated ATP level prompted (Figure 1B),
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