RESUMEN
The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.
Asunto(s)
Disulfuros/metabolismo , Glutatión/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Transporte de Electrón , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Unión Proteica , Pliegue de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
The phosphorylated derivative of the immunosuppressant FTY720 interacts with and modulates the function of sphingosine-1-phosphate (S1P)-receptors. We observed a significant reduction of endothelial surface binding of a S1P(1)-specific antibody after FTY720 treatment of 6h and longer, which was associated with a reduced healing after mechanic injury, impaired angiogenesis and enhanced adhesion molecule expression. FTY720 (5h) had no impact on the expression of S1P(1)- or S1P(3)-encoding transcripts. Notably, pre-treatment of cells with FTY720 for only 30min, which did not reduce S1P(1) surface expression, inhibited the rapid S1P- and SEW2871- (a S1P(1) agonist) induced cortical actin formation and cell migration. FTY720 was effective at concentrations as low as 5nM. FTY720 at therapeutic concentrations may be harmful by impairing important endothelial functions. Interestingly, FTY720 inhibited endothelial actin remodelling and cell migration without decreasing S1P(1) surface expression.