Endogenous S1P is bound to PHB2 in cells. A) HeLa cell lysates were incubated for 30 min with [³²P]S1P (0.1 nM) in the absence or presence of 10 μM unlabeled S1P, dihydro-S1P (DHS1P), sphingosine, or LPA, as indicated. Cell lysates were then immunoprecipitated with antibodies against PHB2 or with isotype-matched IgG antibody, and radioactivity in the immunocomplexes was determined by scintillation counting. Data are expressed as means ± sd. *P < 0.05. B–D) HeLa cell lysates were immunoprecipitated with antibodies against PHB1 or PHB2 or with control IgG, as indicated. Immunocomplexes were captured with protein A/G agarose beads and washed extensively. B) Equal amounts of proteins were analyzed by Western blotting with specific antibodies for PHB1 and PHB2. C, D) Lipids were extracted from duplicate samples, and sphingolipids were analyzed by LC-ESI-MS/MS. C) Extracted ion chromatogram for LC-MS/MS reverse-phase separation of PHB2-specific antibody (red), and control IgG immunoprecipitates (blue). D) Quantification of S1P bound to IgG, PHB1, and PHB2 immunocomplexes. Data are means ± sd. E–G) Interaction of S1P and PHB2 in mitochondria. Mitochondria (mito), cytosol (cyto), and nuclear (nuc) fractions were isolated from HeLa cell lysates by subcellular fractionation. E) Proteins were analyzed by immunoblotting with anti-PHB1 and anti-PHB2 antibodies. Antibodies against subunit IV of cytochrome-c oxidase, COX (IV), lamin, and tubulin were used as specific organelle markers for mitochondria, nuclei, and cytosol, respectively. F) Proteins immunoprecipitated with antibodies against PHB2 or with control IgG were analyzed by immunoblotting with specific antibodies for PHB1 and PHB2. Similar results were obtained in two additional experiments. G) Mitochondria extracts were incubated with control (no lipid), sphingosine (Sph), or S1P affinity matrices, and washed extensively; bound proteins were separated by SDS-PAGE and then analyzed by Western blotting with anti-PHB2 or anti-PHB1 antibodies. Inputs are shown in the leftmost lanes.

SphK2 localized in the mitochondrial inner membrane produces S1P that binds to endogenous PHB2. A) Membrane (MF) and soluble (matrix) fractions (SF) were prepared from wild-type heart mitochondria (mito). B) Mitochondria were treated with trypsin (50 μg/ml) or digitonin (0.05 and 0.5%) for 15 min at 4°C. Equal amounts of proteins were analyzed by Western blotting with the indicated antibodies. C) Heart mitochondria extracts from wild-type and sphk2^−/− mice were immunoprecipitated with anti-PHB2 antibody, and immunocomplexes were analyzed by Western blotting with the indicated antibodies. D) Heart mitochondria extracts from wild-type and sphk2^−/− mice were immunoprecipitated with anti-PHB1 or anti-PHB2 antibodies or control IgG as indicated; lipids were extracted from immunoprecipitates, and S1P was determined by LC-ESI-MS/MS. Data are means ± sd. *P < 0.05. E) Wild-type mitochondria extracts were incubated with S1P, sphingosine (Sph), or control (no lipid) affinity matrices and washed extensively, and bound proteins were separated by SDS-PAGE and then analyzed by Western blotting with anti-PHB2 or anti-PHB1 antibodies. Inputs are shown in the leftmost lanes.

Aberrant interaction between PHB2 and COX in SphK2-null mitochondria. A–C) Analysis of mitochondrial electron transport chain complexes by BN-PAGE. A, B) Equal amounts of heart mitochondrial proteins from wild-type and sphk2^−/− mice were separated by BN-PAGE (A) or transferred to nitrocellulose and immunoblotted with the indicated specific complex antibodies (B). C) In-gel catalytic activity assay for COX using the same gel as in A. C I–V, mitochondrial complexes I–V, respectively. Arrows indicate the additional band present only in sphk2^−/− mitochondria. D, E) Interaction between PHB2 and COX. Mitochondria extracts from wild-type and sphk2^−/− hearts were immunoprecipitated with anti-PHB2 antibody (D) or with an antibody against the holocytochrome-c oxidase complex (E) and then analyzed by Western blotting with the indicated antibodies. Inputs are shown at left. Results are representative of 3 experiments.

SphK2, S1P, and respiration in cardiomyocytes. Cardiomyocytes were prepared from wild-type and sphk2^−/− hearts. A) Equal amounts of lysate proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ns, nonspecific band. B) Lipids were extracted from sphk2^+/+ and sphk2^−/− cardiomyocytes, and mass levels of S1P, dihydro-S1P (DHS1P), sphingosine (Sph), and dihydrosphingosine (DHSph) were determined by LC-ESI-MS/MS. *P < 0.05 vs. wild type. C) Duplicate lysates were incubated with S1P affinity matrices and washed extensively, and bound proteins were separated by SDS-PAGE, then analyzed by Western blotting with anti-PHB2 or anti-PHB1 antibodies. Inputs are shown in the leftmost lanes. D, E) Respiratory complexes I, II, and IV in SphK2-null cardiomyocytes display a decreased rate of oxygen consumption. D) Representative respiration traces of digitonin-permeabilized wild-type (red) and sphk2^−/− (green) cardiomyocytes. Sensitive rates were measured by successively adding substrates and specific inhibitors for complexes I, II, and IV. After stimulation with 2 mM ADP, each complex of sphk2^−/− cardiomyocytes displayed a decreased rate of O₂ consumption. E) Maximum rates of respiration of wild-type and sphk2^−/− cardiomyocytes (n=4) in the presence of 2 mM ADP were measured with respiratory complex I, II, and IV substrates. Data are expressed as mean ± se respiration (nmol O₂ · min⁻¹ · mg protein⁻¹). Similar results were obtained in 3 additional experiments. *P < 0.05 vs. wild type.

SphK2 and PHB2 regulate HeLa cell mitochondrial function. HeLa cells were transfected with siControl, siSphK2, siPHB2, or both, as indicated. A, B) mRNA levels (A) and protein (B) were determined by quantitative PCR and Western blotting, respectively. C) Mitochondria were stained with anti-cyclophilin D antibody and visualized by confocal microscopy with a ×63 objective. Scale bar = 10 μm. D) Transfected HeLa cells were stained with TMRM, and mitochondrial membrane potential was determined by confocal microscopy with a ×40 objective. Data are expressed as mean ± se percentage of siControl. E) Maximum rates of respiration in the presence of 2 mM ADP were measured polarographically with respiratory complex I, II, and IV substrates. Data are expressed as mean ± se respiration (nmol O₂ · min⁻¹ · mg protein⁻¹). Similar results were obtained in 3 additional experiments. *P < 0.05 vs. siControl.

A–C) Identification of PHB2 as an S1P binding protein. A) Control (no lipid) or S1P affinity beads were incubated without or with MCF7 nuclear extracts (NE) and extensively washed; bound proteins were resolved by SDS-PAGE and were visualized with SYPRO Ruby protein gel stain. Arrow indicates the band that was excised and sequenced by mass spectrometry. Dark stained bands that appear in the no extract lane are from the affinity matrix. B) Bound proteins from duplicate nuclear extracts were resolved by SDS-PAGE and analyzed by Western blotting with anti-PHB2 antibody. C) Amino acid sequence of PHB2. Red indicates peptide sequences identified by mass spectrometry. D–F) PHB2 specifically binds S1P. D) Nuclear extracts from MCF7 cells were incubated with control (no lipid), LPA, or S1P affinity matrices, as indicated, and washed extensively; bound proteins were separated by SDS-PAGE and then analyzed by Western blotting with anti-PHB2 or anti-PHB1 antibodies. Inputs are shown in the leftmost lanes. E) Nuclear extracts were preincubated without or with 10 μM S1P for 2 h, followed by pulldown with S1P beads. Beads were washed, and bound proteins were analyzed by Western blotting with anti-PHB2 or anti-PHB1 antibodies. F) Nuclear extracts from MCF7 cells were incubated for 30 min with [³²P]S1P (0.1 nM) in the absence or presence of 10 μM unlabeled S1P or sphingosine. Cell lysates were then immunoprecipitated with antibodies against PHB1 or PHB2 or with control IgG, and radioactivity in the immunocomplexes was determined by scintillation counting. Data are expressed as means ± sd. *P < 0.05.

SphK2 and S1P in cardiac mitochondria. A) Mitochondria (mito), and nuclear fractions were prepared from wild-type and sphk2^−/− hearts. Equal amounts of proteins were separated by SDS-PAGE and immunoblotted with anti-SphK2, anti-PHB2, and anti-PHB1 antibodies. Antibodies against subunit IV of cytochrome-c oxidase, COX (IV), and VDAC2 or lamin were used as specific organelle markers for mitochondria and nuclei, respectively, and to show equal loading and transfer. ns, nonspecific bands. B) SphK1 and SphK2 activities were determined by isotype-specific assays (30) in extracts of mitochondria isolated from hearts of wild-type and SphK2-knockout mice. C) Lipids were extracted from mitochondria isolated from sphk2^+/+ and sphk2^−/− hearts and mass levels of sphingoid bases sphingosine (Sph) and dihydrosphingosine (DHS), and their phosphorylated products, S1P and dihydro-S1P (DHS1P), ceramide (Cer), and sphingomyelin (SM) were determined by LC-ESI-MS/MS. Data are expressed as means ± sd. *P < 0.01 vs. wild type.