Respiratory state and phosphatidylserine import in brain mitochondria in vitro

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized s tate was monitored by flow cytometry measuring the fluorescence of membrane -potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association wi th liposomes. After interaction of mitochondria with C-14-PS containing lip osomes, C-14-PS became a substrate of PS decarboxylase, as monitored by the formation of C-14-phosphatidylethanolamine (PE), indicating translocation of C-14- PS to the inner membrane. The kinetics of C-14-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared t o control (state I). In state 3,C-14-PE formation decreased in the presence of NaN3. Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria h as not been completely elucidated. A crude mitochondrial fraction (P-2) con taining MAM, synaptosomes and myelin was prelabeled with C-14-PS and incuba ted in different respiratory states. At a high respiratory rate, low-densit y labeled mitochondria, whose band overlaps that of synaptosomes, were obta ined by centrifugation. A parallel decrease of both radioactivity and prote in in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of C-14-PS in P-2 m embranes were also studied by incubating P-2, with C-14-serine. In the rest ing state C-14 PS accumulated in MAM, indicating that the transfer to mitoc hondria was a limiting step. In state 3 both the transfer rate of C-14-PS a nd its conversion to C-14-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism t hat allowed the transport of PS across the outer mitochondrial membrane.