Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine.
A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.
Emoto
K
K
Department of Molecular Biodynamics, The Tokyo Metropolitan Institute of Medical Science (RINSHOKEN), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Kuge
O
O
Nishijima
M
M
Umeda
M
M
D000818:Animals; D001692:Biological Transport; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000378:metabolism; D016466:CHO Cells; D002262:Carboxy-Lyases / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D008928:Mitochondria / Q000378:metabolism*; D009154:Mutation; D010714:Phosphatidylethanolamines / Q000096:biosynthesis; D010718:Phosphatidylserines / Q000378:metabolism*
D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase