Figure 4.

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.