Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells

The synthesis of phosphatidylserine and its translocation to the mitochondria were examined in permeabilized Chinese hamster ovary (CHO)-K1 cells by following the metabolism of a [3H]serine precursor to [3H] phosphatidylserine (PtdSer) and [3H]phosphatidylethanolamine (PtdEtn). In physiological salt solutions approximating the intracellular ionic composition, both the synthesis of PtdSer and its translocation required ATP. The ATP requirement for PtdSer synthesis could be completely bypassed, and that for translocation could be partially bypassed at Ca2+ concentrations 10(3)-10(4) times the intracellular physiological level (i.e. 1 mM). The ATP-dependent synthesis of PtdSer could be inhibited by chelation of Ca2+ with EGTA, inhibition of Ca2+ sequestration with 2,5-di(tert-butyl)hydroquinone, mobilization of sequestered Ca2+ with ionomycin, and competition for [3H]serine with ethanolamine. The inhibition of the ATP-dependent synthesis of PtdSer by the aforementioned inhibitors provided an efficient method to rapidly arrest the incorporation of [3H]serine into [3H]PtdSer. By pulse-labeling the [3H]PtdSer pool and arresting further synthesis with inhibitors, the translocation of nascent PtdSer could be uncoupled from synthesis. The results of these pulse-labeling-arrest experiments provide unambiguous evidence that PtdSer translocation to the mitochondria is not driven by PtdSer synthesis. The addition of apyrase to ATP-supplemented, permeabilized cells abruptly terminates [3H]serine incorporation into [3H]PtdSer and the decarboxylation of [3H]PtdSer to [3H]PtdEtn, thereby demonstrating that a specific ATP requirement exists for the translocation of nascent PtdSer to the mitochondria in permeabilized cells. The translocation of nascent PtdSer to the mitochondria was unaffected by 45-fold dilution of the standard reaction thus indicating that the translocation intermediate was unlikely to be a freely diffusible complex. The requirements for translocation of nascent phosphatidylserine are different from those for the vesicular movement of proteins insofar as the lipid movement does not require cytosol and is unaffected by the addition of Ca2+, GTP, or GTP gamma S. From these studies, we conclude that: 1) the synthesis and translocation of PtdSer can be readily studied in permeabilized cells, 2) the ATP-dependent synthesis of PtdSer is functionally coupled to the ATP-dependent sequestration of Ca2+ by the endoplasmic reticulum or closely related membranes, 3) PtdSer translocation is independent of its synthesis, and 4) there is a specific requirement for ATP in the translocation of PtdSer to the mitochondria.