Import of phosphatidylethanolamine for the assembly of rat brain mitochondrial membranes

Mitochondria can synthesize phosphatidyl-ethanolamine (PE) through phosphatidylserine decarboxylase (PS decarboxylase) activity or can import this lipid from the endoplasmic reticulum. In this work, we studied the factors influencing the import of PE in brain mitochondria and its utilization for the assembly of mitochondrial membranes. Incubation of rat brain homogenate with [1-3H]ethanolamine resulted in the synthesis and distribution of 3H-PE to subcellular fractions. Twenty-one percent of labeled PE was recovered in purified mitochondria. The import of PE in mitochondria was studied in a reconstituted system made of microsomes (donor particles) and purified mitochondria (acceptor particles). Ca2+ and nonspecific lipid transfer protein purified from liver tissue (nsL-TP) enhanced the translocation process. 3H-PE synthesized in membrane associated to mitochondria (MAM) could also translocate to mitochondria in the reconstituted system. Exposure of mitochondria to trinitrobenzensulfonic acid (TNBS) resulted in the reaction of more than 60% of 3H-PE imported from endoplasmic reticulum and of about 25% of 14C-PE produced in mitochondria by decarboxylation of 14C-PS. Moreover, the removal of the outer mitochondrial membrane by digitonin treatment, resulted in the loss of 3H-PE, but not 14C-PE. These results indicate that labeled PE imported in mitochondria is mainly localized in the outer mitochondrial membrane, whereas PE produced by PS decarboxylase activity is confined to the inner mitochondrial membrane. Phospholipase C hydrolyzed 25-30% of both PE radioactivity and mass of the outer mitochondrial membrane indicating an asymmetrical distribution of this lipid across the membrane.