Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 15;288(7):5176-85.
doi: 10.1074/jbc.M112.413617. Epub 2013 Jan 4.

Identification of a Mammalian-Type Phosphatidylglycerophosphate Phosphatase in the Eubacterium Rhodopirellula Baltica

Affiliations
Free PMC article

Identification of a Mammalian-Type Phosphatidylglycerophosphate Phosphatase in the Eubacterium Rhodopirellula Baltica

Phildrich G Teh et al. J Biol Chem. .
Free PMC article

Abstract

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.

Figures

FIGURE 1.
FIGURE 1.
Identification of a putative PTPMT1 ortholog in R. baltica. A, schematic diagram of the CL biosynthesis pathway. Enzymes are highlighted in blue. CDP-DAG, cytidine diphosphate diacylglycerol; G3P, glycerol-3-phosphate. B, active site primary sequence alignment of PTPMT1 orthologs using PROMALS3D and illustrated by ESPript (49, 50). H. sap (H. sapiens), NP_783859; D. mel (D. melanogaster), NP_732901; and R. balt (R. baltica), NP_865112. C, a phylogenetic tree of human and bacterial DSPs constructed using maximum likelihood (ML) method in PHYML. Each branch was tested by 100 bootstrap replicates, and only branches with bootstrap values above 50% were shown. Human PTPMT1 is bolded and bacterial PTPMT1 orthologs are highlighted in red. D, domain architecture of PGP phosphatases. All domains are presented based on analyses using PFAM, CDD, and PROSITE. Catalytic active site sequences are indicated above. MTS, mitochondrial targeting sequence; DSP, dual specificity phosphatase; HAD, haloacid dehalogenase; PAP2, phosphatidic acid phosphatase 2. E, presence and absence matrix for CL de novo synthesis enzymes in Rhodeopirellula. Dark gray squares indicate presence of CL enzyme (column) in target species (row). Peach squares mean no ortholog has been identified, i.e. blast search failed to identify sequences that meet the bidirectional-best-hit criterion (cut-off E-values of 10−10 or less). Sequences of E. coli pgsA, S. cerevisiae GEP4, H. sapiens PTPMT1, E. coli pgpA, pgpB, pgpC, and E. coli cls1 were used as queries for the blast searches.
FIGURE 2.
FIGURE 2.
Analysis of bacterial PTPMT1 activity in vitro. A, specific activities of recombinant murine (M. mus), fly (D. mel), and bacterial (R. balt) PTPMT1 are measured using pNPP as substrate. Data were calculated from the change in absorbance at 410 nm and represented as the mean ± S.D. of triplicate measurements. CS, catalytically inactive mutant. B, synthesis of 14C-PGP. Highlighted in blue is the phosphate group cleaved by PTPMT1 and marked with red asterisks are the radiolabled carbons. C, activities of wild-type and catalytically inactive PTPMT1 orthologs against PGP. Lipid products from the reactions were separated by TLC and analyzed by autoradiography. A trace amount of PGP phosphatase activity is seen in the background, because a contaminant E. coli PGP phosphatase co-purified with the recombinant PGS proteins (18). D, phosphatase assay conditions are determined for each indicated enzyme by measuring the amount of free phosphate released using the malachite green reagent. The reaction was carried out at 30 °C and in the presence of 100 μm PGP. Enzyme concentrations are indicated in the insets. E, comparison of enzymatic activities. Phosphatase activity was calculated under initial linear rate conditions (30 °C, 100 μm PGP, and 30 s of incubation time), compared with that of M. mus PTPMT1, and represented as the mean ± S.D. from three independent experiments. F, saturation curve of the Rhodopirellula PTPMT1 ortholog. The phosphatase assay was carried out under initial linear rate conditions and in the presence of various PGP concentrations. A nonlinear regression analysis was performed to indicate the saturation. G, effect of Triton X-100 on Rhodopirellula PTPMT1 PGP phosphatase activity.
FIGURE 3.
FIGURE 3.
Bacterial PTPMT1 functionally compensates for loss of yeast gep4 in vivo. A, wild-type murine, fly, and bacterial PTPMT1 complement the growth deficiency of GEP4-null cells. The expression levels of FLAG-tagged PTPMT1s and GEP4 are indicated by Western blot analysis. YPD, yeast extract-peptone-glucose medium; SCD, synthetic completed medium with dextrose; EtBr, ethidium bromide. CDC2 levels are shown as the loading control. Detection of steady state PGP (B) and CL (C) levels in gep4Δ yeast cells complemented with control plasmid or plasmids encoding PTPMT1 orthologs. Cells were labeled with [32P]orthophosphate at 10μCi/ml for 12 h. Lipids were extracted, separated via TLC, and viewed by autoradiography. D, PGP and CL contents are determined by the ratio of 32P incorporated into PGP versus total phospholipids, and represented as the mean ± S.D. from three independent experiments. **, p ≤ 0.01; *, p ≤ 0.05.
FIGURE 4.
FIGURE 4.
Mitochondrial localization of the Rhodopirellula PTPMT1. A, N-terminal sequences of PTPMT1 orthologs are aligned using PROMALS3D. Amino acid similarities are shaded in light gray and identities in dark gray. The MTS of mouse PTPMT1 has been previously characterized (8) and is indicated by the blue bar. B, N-terminal region of bacterial PTPMT1 forms an amphiphilic helix. 18 amino-terminal residues were plotted on the helical wheel using HeliQuest (51). Basic residues are colored blue, and hydrophobic residues are colored yellow. C, subcellular fractionation of yeast gep4Δ cells expressing fly or bacterial PTPMT1. 30 μg of protein from each fraction were analyzed by SDS-PAGE and immunoblotting. Porin, Calreticulin, and PGK1 serve as marker proteins for mitochondria, ER, and cytosol, respectively. D, bacterial PTPMT1 localizes to the mitochondria of mammalian COS-1 cells. 24 h post-transfection, cells were stained with MitoTracker Red, fixed, and analyzed by immunofluoresence imaging.

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Hunter T. (1987) A thousand and one protein kinases. Cell 50, 823–829 - PubMed
    1. Alonso A., Sasin J., Bottini N., Friedberg I., Osterman A., Godzik A., Hunter T., Dixon J., Mustelin T. (2004) Protein tyrosine phosphatases in the human genome. Cell 117, 699–711 - PubMed
    1. Tonks N. K., Neel B. G. (2001) Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13, 182–195 - PubMed
    1. Fauman E. B., Saper M. A. (1996) Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci 21, 413–417 - PubMed
    1. Tagliabracci V. S., Turnbull J., Wang W., Girard J. M., Zhao X., Skurat A. V., Delgado-Escueta A. V., Minassian B. A., Depaoli-Roach A. A., Roach P. J. (2007) Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo. Proc. Natl. Acad. Sci. U.S.A. 104, 19262–19266 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback