The potent lipid mediator sphingosine-1-phosphate (S1P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S1P, produced in the mitochondria mainly by sphingosine kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S1P did not bind to the closely related protein PHB1, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2-null mice, a new aberrant band of cytochrome-c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome-c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome-c oxidase. Our data point to a new action of S1P in mitochondria and suggest that interaction of S1P with homomeric PHB2 is important for cytochrome-c oxidase assembly and mitochondrial respiration.

Background-Opening of the mitochondrial permeability transition pore (mPTP) is a crucial event in lethal reperfusion injury. Phosphorylation (inhibition) of glycogen synthase kinase-3␤ (GSK3␤) has been involved in cardioprotection.We investigated whether phosphorylated GSK3␤ may protect the heart via the inhibition of mPTP opening during postconditioning. Methods and Results-Wild-type and transgenic GSK3␤-S9A mice (the cardiac GSK3␤ activity of which cannot be inactivated) underwent 60 minutes of ischemia and 24 hours of reperfusion. At reperfusion, wild-type and GSK3␤-S9A mice received no intervention (control), postconditioning (3 cycles of 1 minute ischemia and 1 minute of reperfusion), the mPTP inhibitor cyclosporine A (CsA; 10 mg/kg IV), or the GSK3␤ inhibitor SB216763 (SB21; 70 g/kg IV). Infarct size was assessed by triphenyltetrazolium chloride staining. The resistance of the mPTP to opening after Ca 2ϩ loading was assessed by spectrofluorometry on mitochondria isolated from the area at risk. In wild-type mice, infarct size was significantly reduced by postconditioning, CsA, and SB21, averaging 39Ϯ2%, 35Ϯ5%, and 37Ϯ4%, respectively, versus 58Ϯ5% of the area at risk in control mice (PϽ0.05). In GSK3␤-S9A mice, only CsA, but not postconditioning or SB21, reduced infarct size. Postconditioning, CsA, and SB21 all improved the resistance of the mPTP in wild-type mice, but only CsA did so in GSK3␤-S9A mice. Conclusion-These