Phosphatidylserine synthase-1 and-2 are localized to mitochondria-associated membranes

We report the subcellular localization of enzymes involved in phosphatidyls erine biosynthesis in mammalian cells, Several lines of evidence suggest th at phosphatidylserine synthase-l (PSS1) is highly enriched in mitochondria- associated membranes (MAM) and is largely excluded from the bulk of the end oplasmic reticulum (ER). Taking advantage of the substrate specificity of P SS1, we showed that (i) MAM contain choline exchange activity, whereas this activity is very low in the bulk of the ER, (ii) serine exchange activity is inhibited by choline to a much greater extent in MAM than in ER, and (ii i) MAM use phosphatidylcholine and phosphatidylethanolamine as substrates f or phosphatidylserine biosynthesis, whereas the ER utilizes only phosphatid ylethanolamine. According to immunoblotting of proteins from both CHO-K1 ce lls and murine liver, PSS1 is localized to MAM, and in hepatoma cells stabl y expressing PSS1 this protein is highly enriched in MAM. Since the ER cont ains serine and ethanolamine exchange activities, we had predicted that PSS 2 would account for the serine exchange activity in the ER. Unexpectedly, u sing immunoblotting experiments, we found that (i) PSS2 of CRO-K1 cells is present only in MAM and (ii) PSS2 is restricted to MAM of McArdle cells exp ressing recombinant PSS2. These data leave open the question of which enzym e imparts PSS activity to the ER and suggest that a third isoform of PSS mi ght be located in the ER.