A MITOCHONDRIAL-MEMBRANE PROTEIN IS REQUIRED FOR TRANSLOCATION OF PHOSPHATIDYLSERINE FROM MITOCHONDRIA-ASSOCIATED MEMBRANES TO MITOCHONDRIA

The mechanism of import of phosphatidylserine (PtdSer) into mitochondr ia was investigated using a reconstituted system of isolated organelle s in vitro in which PtdSer was translocated from donor membranes to mi tochondria and was decarboxylated. therein. Neither phosphatidylcholin e nor phosphatidylethanolamine (PtdEtn) was translocated under the sam e conditions. Transfer of PtdSer from its site of synthesis on the end oplasmic reticulum and mitochondria-associated membranes [J. E. Vance (1990) J. Biol. Chem. 265, 7248-7256] to its site of decarboxylation o n mitochondrial inner membranes is predicted to be mediated by membran e contact. A mitochondrial membrane protein appears to be involved in the translocation event since proteolysis of proteins exposed on the m itochondrial surface potently inhibited PtdSer transfer, whereas prote olysis of surface proteins of mitochondria-associated membranes did no t impair the transfer. The nature of the membranes that donate PtdSer to mitochondria in vitro is not crucial since PtdSer of mitochondria-a ssociated membranes, endoplasmic reticulum and microsomes was decarbox ylated to PtdEtn with approximately equal efficiency. The translocatio n of PtdSer to mitochondria was stimulated by magnesium and calcium io ns and was inhibited by incubation of mitochondria with sulphydryl gro up-modifying reagents. Reconstitution of PtdSer translocation/decarbox ylation using digitonin-solubilized mitochondria and PtdSer-donor memb ranes suggested that the putative PtdSer-translocation protein is prim arily localized to contract sites between mitochondrial inner and oute r membranes. These studies provide evidence for the involvement of a m itochondrial membrane protein in the import of newly-synthesized PtdSe r into mitochondria.