Characterization of phosphatidylserine transport to the locus of phosphatidylserine decarboxylase 2 in permeabilized yeast

In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mit ochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidyle thanolamine (PtdEtn). In strains with a psd1 Delta allele for the mitochond rial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can s erve as an indicator of lipid transport to the locus of PtdSer decarboxylas e 2 (Psd2p) in the Golgi/vacuole. We have followed the metabolism of [H-3]s erine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1 Delta yeast. The permeabilized cells synthesize H-3-PtdSer and, after a 20 -min lag, decarboxylate it to form [H-3]PtdEtn, Formation of [H-3]PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing P tdSer synthesis. PtdSer transport can be resolved into a two-component syst em using washed, permeabilized psd1 Delta cells as donors and membranes iso lated by ultracentrifugation as accepters, With this system, the transport- dependent decarboxylation of nascent PtdSer is dependent upon the concentra tion of acceptor membranes, requires Mn2+ but not nucleotides, and is inhib ited by EDTA High speed membranes isolated from a previously identified Ptd Ser transport mutant, pstB2, contain normal Psd2p activity but fail to reco nstitute PtdSer transport and decarboxylation, Reconstitution with permutat ions of wild type and pstB2 Delta donors and accepters identifies the site of the mutant defect as the acceptor side of the transport reaction.