PLASMA-MEMBRANE TRANSLOCATION OF FLUORESCENT-LABELED PHOSPHATIDYLETHANOLAMINE IS CONTROLLED BY TRANSCRIPTION REGULATORS, PDR1 AND PDR3

The transcription regulators, PDR1 and PDR3, have been shown to activa te the transcription of numerous genes involved in a wide range of fun ctions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We rep ort here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-la beled (M-C-6-NBD-PE) phosphatidylethanolamine across the plasma membra ne, A combination of fluorescence microscopy, fluorometry, and quantit ative analysis demonstrated that M-C-6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants , defective in the accumulation of M-C-6-NBD-PE, were isolated by sele ctively photokilling normal cells that accumulated the fluorescent pho spholipid, This led to the isolation of numerous trafficking in phosph atidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C-6-NBD-PE, Complementation cloning and linkage anal ysis led to the identification of the dominant mutation TPE1-1 as a ne w allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3. The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labe ling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant st rains increased four- to fivefold relative to the parent Tpe(+) strain , indicating that the net inward flux of endogenous phosphatidylethano lamine as well as M-C-6-NBD-PE was decreased, Targeted deletions of PD R1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, re sulted in normal M-C-6-NBD-PE accumulation, confirming that PDR1-11 an d pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respective ly. Both mutant alleles resulted in resistance to the drugs cyclohexim ide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a prev iously identified drug-resistant allele, pdr3-2, accumulated normal am ounts of M-C-6-NBD-PE, indicating allele specificity for the loss of M -C-6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 r egulate the net rate of M-C-6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine acro ss the plasma membrane.