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.