Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells.
Chinese hamster ovary (CHO-K1) cells were pulse labeled with [3H]serine, and the synthesis of phosphatidyl[3H]ethanolamine from phosphatidyl[3H]serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 micrograms of saponin per ml, there was no significant turnover of nascent phosphatidyl[3H]serine to form phosphatidyl[3H]ethanolamine during an ensuing chase. Saponin treatment rendered greater than 99% of the cells permeable as judged by trypan blue exclusion and depleted them of 85% of their complement of cytosolic proteins as determined by residual lactic acid dehydrogenase activity. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl[3H]ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl[3H]serine during a subsequent 2-hr chase. Phosphatidyl[3H]ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl[3H]ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl[3H]serine to phosphatidyl[3H]ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5'-adenylyl imidodiphosphate, or apyrase (2.5 units/ml) plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl[3H]ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.
Voelker
D R
DR
Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002262:Carboxy-Lyases / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000096:biosynthesis; D010718:Phosphatidylserines / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D012503:Saponins
D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D012265:Ribonucleotides; D012503:Saponins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase