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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 activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane. A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid. This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE. Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new 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 labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased. Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively. Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane.

thought to be responsible for establishing their asymmetric distribution, it is not known how many phospholipid translocases are required or how they are regulated to establish the appropriate phospholipid distribution.
In this paper, we developed a genetic approach using the yeast Saccharomyces cerevisiae to address these questions. First, we characterized the influx and efflux pathways for 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) 1 -labeled phosphatidylethanolamine (M-C 6 -NBD-PE) and found that influx occurred by a mechanism that has similar characteristics to the flippase-mediated uptake of phosphatidylethanolamine in mammalian cells. Second, we developed a novel method of mutant enrichment by dye-sensitized photokilling and isolated new mutant alleles in two genes ( PDR1 and PDR3 ) that result in the loss of M-C 6 -NBD-PE accumulation in cells and an increase in the amount of endogenous phosphatidylethanolamine in the outer leaflet of the plasma membrane. Both PDR1 and PDR3 encode transcription factors that activate the transcription of numerous genes that confer resistance to a wide range of drugs (5). The results indicated that PDR1 and PDR3 regulate phosphatidylethanolamine distribution across the plasma membrane, presumably by increasing the transcription of one or more undetermined genes that regulate the net translocation (flip-flop) of phosphatidylethanolamine across the plasma membrane.

Materials
Yeast media were obtained from Difco Laboratories (Detroit, MI). Restriction enzymes and molecular biology reagents were purchased from Life Technologies (Gaithersburg, MD). Unless otherwise noted, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Yeast Strains and Culture
The Saccharomyces cerevisiae strains used are shown in Table I. For all experiments, early to mid-log phase cultures (OD 600 ϭ 0.2-1) were grown from either overnight cultures or fresh plates in the indicated media as described in Sherman et al. (44). The media used were: YPD, medium containing 1% yeast extract, 2% glucose, 2% peptone; YEP-GE, medium containing 1% yeast extract, 2% peptone, 2% glycerol, and 2% ethanol; YP-Gal, medium containing 1% yeast extract, 2% galactose, 2% peptone; SDC, synthetic complete media: 0.67% yeast nitrogen base, 2% glucose, complete amino acid supplements; SCNaN 3 , SDC lacking glucose but containing 2% sorbitol and 20 mM sodium azide. Temperature-sensitive strains were grown at a permissive temperature of 23 Њ C, and internalization assays were performed at the nonpermissive temperature of 37 Њ C. Yeast transformations were performed as described by Gietz et al. (19).

Vesicle Preparation
M-C 6 -NBD-PE, M-C 6 -NBD-PC, dioleoylphosphatidylcholine (DOPC), and N -rhodamine-dioleoylphosphatidylethanolamine ( N -Rh-DOPE) were from Avanti Polar Lipids Inc. (Alabaster, AL). FM4-64 was purchased from Molecular Probes, Inc. (Eugene, OR). Phospholipids were stored at Ϫ 20 Њ C, periodically monitored for purity by thin-layer chromatography (TLC), and repurified when necessary. Phospholipid concentrations were determined by lipid phosphorus assay (2). To prepare vesicles, lipids were first mixed in desired proportions, and the chloroform was removed by evaporation under nitrogen followed by overnight vacuum desiccation. Desiccated lipids were then solubilized either in SDC medium or SDC medium lacking glucose (for ATP-depletion experiments), and the mixture was passed eight times through a Lipex Extruder (Lipex Biomembranes, Inc., Vancouver, BC, Canada) equipped with 0.1-mm filters to produce vesicles. Total lipid concentration in the stock vesicle preparation was 1 mM. Typically, proportions were 40 mol% NBD-labeled phospholipid, 2 mol% N -Rh-DOPE, and 58 mol% DOPC.

Internalization of Fluorescent Lipids into Yeast Cells
Cells were grown to mid-log phase in YPD or SDC at 30 Њ C. Cells were washed three times in SDC and resuspended at ف 10 7 cells/ml. Donor vesicles containing 40% NBD-labeled phospholipid, 58% DOPC, and 2% N -Rh-DOPE (50 M total lipid concentration) were added to the cells and incubated for 30-120 min. Cells were washed three times with ice-cold SCNaN 3 before microscopy. Specific incubation conditions for the different fluorescent lipids are as follows: Internalization experiments with M-C 6 -NBD-PE typically entailed incubation with donor vesicles for 30 min before microscopy. Experiments with M-C 6 -NBD-PC typically entailed incubation for 2 h with the fluorescent lipid before microscopy. FM4-64 internalization experiments were performed by adding 1 l of this dye from a 1 mg/ml ethanolic stock solution to 1 ml of cells at ف 1 ϫ 10 7 /ml and continuing incubation for 2 h before microscopy.
For M-C 6 -NBD-PE internalization experiments with sec mutants, growth to mid-log phase was carried out at 23 Њ C. Cultures were then split, and half of the cells were warmed to the restrictive temperature of 37 Њ C, while the other half remained at 23 Њ C. Warming to 37 Њ C was accomplished by gradually increasing the temperature over a period of ف 10 min. After shifting the temperature for 30 min, vesicles were added, and incubation at 37 Њ C was continued for 1 h. Cells were then transferred to an ice-water bath and washed rapidly with ice-cold SCNaN 3 medium in preparation for microscopy. Controls were processed identically.
To quantitate the results with the sec mutants and their isogenic Sec ϩ strains, 15 to 30 cells were analyzed for each of the strains at their permissive (23 Њ C) and nonpermissive (37 Њ C) temperatures for growth. Although there were strain to strain variations in M-C 6 -NBD-PE accumulation at the permissive temperature for each isogenic pair, the difference in accumulation at 23 Њ C did not exceed 27%. The temperature-sensitive accumulation of M-C 6 -NBD-PE for each strain was expressed as a ratio of the average pixel brightness at 37 Њ C relative to 23 Њ C. Inhibition of growth of the sec mutants at 37 Њ C was used to confirm the temperature-sensitive growth phenotype of all of the sec strains used in these studies.
To deplete intracellular ATP, cells were incubated with shaking in SCNaN 3 for 45-50 min at 30 Њ C before labeling as described above. We have previously shown that this treatment results in 80% reduction in cellular ATP levels (26). To test the effect of N -ethylmaleimide (NEM) treatment on M-C 6 -NBD-PE accumulation, mid-log phase cells were cooled to 2 Њ C, harvested by centrifugation, and resuspended in buffered media (SDC containing 50 mM sodium phosphate buffer, pH 7.5) containing 2 mM NEM. Cells were incubated on ice for 30 min and were shaken after the first 15 min. Cells recovered by centrifugation were resuspended in buffered medium containing 2 mM DTT and incubated on ice for 5 min. Cells were pelleted again, washed once with buffered medium, and resuspended in buffered medium before labeling with M-C 6 -NBD-PE as described above. After labeling, cells were incubated on ice for 1.5 h and washed three times with ice-cold SCNaN 3 buffered with 50 mM sodium phosphate, pH 7.5, before fluorescence microscopy.

Fluorescence Microscopy
Fluorescence microscopy was performed on a microscope (model Axiovert; Carl Zeiss, Inc., Thornwood, NY) equipped with barrier filters that allowed no detectable crossover of NBD and rhodamine fluorescence. For localization of 4 Ј ,6-diamidino-2-phenylindole (DAPI) fluorescence, barrier filters were used that did not allow detectable crossover of NBD and DAPI fluorescence. The fluorescence image was enhanced with an imageintensifying camera (model SIT 66 or VE1000-SIT; DAGE-MTI, Inc., Michigan City, IN), digitized, and stored. Contrast enhancement and pixel brightness analysis of the digital images were performed with Image-1/AT or Metamorph software (Universal Imaging Corp., West Chester, PA).
Average pixel brightness was measured from a rectangle (1 ϫ 3 pixels) in the middle of vacuoles (for vacuolar labeling with M-C 6 -NBD-PC) or a rectangle surrounding the entire cell (for all other labeling experiments). Pixel brightness analysis of DOPC vesicles containing known amounts of M-C 6 -NBD-PC confirmed that the measurements fell within the linear range of the SIT camera. To confirm that the pixel brightness measurements accurately reflected the amount of internalized M-C 6 -NBD-PE, labeled cells were extracted (22) and analyzed by TLC and fluorometry (26). Fluorescence readings of the total extract as well as the TLC-purified M-C 6 -NBD-PE were proportional to the pixel brightness measurements.

Trinitrobenzenesulfonic Acid Labeling
Cells were grown 18 h at 30ЊC to mid-log phase (OD 600 ϭ 0.4-0.7) in SDC containing 500 Ci of [ 32 P]KH 2 PO 4 (specific activity 1 Ci/mmol; New England Nuclear, Boston, MA). Plasma membrane, outer leaflet phosphatidylethanolamine was labeled with TNBS as described previously (30). Cells were harvested by centrifugation, washed twice in ice-cold 40 mM NaCl, 120 mM NaHCO 3 , pH 8.4, resuspended in the same buffer containing 5 mM trinitrobenzenesulfonic acid (TNBS) (Sigma Chemical Co.), and immediately placed on ice for 1 h with periodic vortex mixing. After TNBS labeling, cells were washed by centrifugation three times in fresh buffer, pelleted, and disrupted by vortexing with glass beads. Cellular lipids were extracted with chloroform/methanol (2:1) and separated by twodimensional TLC (first solvent: chloroform/methanol/ammonium hydroxide, 65:35:5; second solvent: chloroform/methanol/acetone/acetic acid/water; 50:10:20:10:5). Spots were identified by comparison with known standards. The percentages of phosphatidylethanolamine and TNP-phosphatidyleth-anolamine were quantified by phosphorimaging with a PhosphorImager SI scanning instrument (Molecular Dynamics, Sunnyvale, CA). Percent viability was determined by counting the number of cells labeled after dilution into 0.02% methylene blue with a hemocytometer.

Isolation of Trafficking in Phosphatidylethanolamine (tpe) Mutants
Isogenic strains LKY6-2B (MATa) and CRY2 (MAT␣) were mutagenized at 23ЊC with ethylmethanesulfonate to 80% killing (27). For dye-sensitized photokilling, mutagenized cells were grown to mid-log phase in SDC at 23ЊC before shifting to 37ЊC for 30 min. Donor vesicles (50 M total lipid concentration) containing 40 mol% M-C 6 -NBD-PE were then added as described above, and incubation continued for 30 min. After the initial 30-min incubation, cells (7 ml at 1 ϫ 10 7 cells/ml) were transferred to a 60-mm Petri dish, which was placed in a chamber equilibrated to 37ЊC by a circulating water bath, and incubation was continued for an additional 2 h (in the continuous presence of vesicles). During this incubation, cells were irradiated with the light from a 150-W xenon lamp (Second Source Inc., LaVerne, CA) that was passed through a narrow band-pass filter at 470-480 nm, producing an intense blue light. After irradiation, cells were washed three times in SDC and either plated for single colonies or incubated overnight at 23ЊC in preparation for successive rounds of photokilling. The temperature shift from 37ЊC (incubation with vesicles and irradiation) to 23ЊC (growth overnight) allowed the potential isolation of temperature-sensitive mutants. The tpe mutants were isolated after five rounds of dye-sensitized photokilling.

Drug Sensitivity Assays
1 mg/ml stock solutions of drugs were made by dissolving drugs in DMSO or ethanol. Resistance to drugs such as cycloheximide, oligomycin, and 4-nitroquinoline-N-oxide was determined using the spot test assay. For the spot test assay, 5-l aliquots of yeast mid-log phase cultures (OD 600 ϭ 0.25) were spotted onto YPD plates containing the indicated concentrations of cycloheximide or 4-nitroquinoline-N-oxide, or YPG plates containing the indicated concentrations of oligomycin. Plates were incubated for 3 d at 23, 30, or 37ЊC.

Cloning of TPE2
The TPE2 gene was identified by its ability to complement the temperature-sensitive growth defect of tpe2-1 on nonfermentable carbon sources. The tpe2-1 strain LKY161 was transformed with the yeast genomic library, YPH1, which contains the selectable marker LEU2. Candidate TPE2 clones were identified based on their ability to restore growth of tpe2-1 on SC-glycerol-ethanol plates lacking leucine at 37ЊC. Only one plasmid was isolated that was able to complement both the M-C 6 -NBD-PE accumulation defect as well as the temperature-sensitive growth defect on nonfermentable carbon sources of tpe2-1. This plasmid was designated p201. p201 contained a 15-kb insert that sequencing revealed was a portion of chromosome II spanning half of the SLA1 gene through approximately one third of the ORF YBL004. Only two full-length ORFs are contained within this region, PDR3 and an uncharacterized ORF YBL006.
To identify the portion of p201 responsible for complementing tpe2-1, LKY161 was transformed with subclones of p201 containing either YBL006 or PDR3. Integrative transformation with YBL006 did not complement tpe2-1; however, transformation with PDR3 resulted in full complementation of both the M-C 6 -NBD-PE accumulation defect as well as the temperature-sensitive growth defect on nonfermentable carbon sources. The strain transformed with PDR3 was designated AGY124.
Linkage analysis was used to determine whether the PDR3 was TPE2 (38). AGY124 was crossed to AGY10, a strain that is wild-type at the TPE2 and LEU2 loci. All spores from this cross were wild-type for the PE accumulation phenotype. This confirmed pAG3 integrated at the PDR3 locus and that TPE2 was linked to PDR3.

Disruption of PDR1 and PDR3
For disruption of the PDR3 gene, a 2.3-kb portion of the PDR3 coding region was removed by digestion with MunI and EcoRI and replaced with the HisG-URA3-HisG cassette of the vector pNKY51 (1). To disrupt PDR1, 2.2 kb of the coding region was removed by digestion with MunI and replaced by the HisG-URA3-HisG cassette. Proper integration of the disrupted alleles was confirmed by PCR or Southern blotting analysis.

Cloning and Sequencing of Mutant and Wild-Type PDR3 Alleles
PDR3 alleles were rescued using the method described by Winston et al. (55). The alleles were obtained by integrative transformation of a YIp vector near the PDR3 locus and subsequent eviction of a plasmid containing the vector along with the full-length PDR3 allele of interest. Plasmid DNA was isolated from Escherichia coli transformants and the presence of PDR3 alleles was confirmed by restriction digest analysis.
The nucleotide sequence of the entire open reading frame along with an additional 400 bp upstream of the translation initiation codon and 250 bp downstream of the termination codon of each PDR3 allele was determined. Sequencing reactions were performed using the PRISM Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA), an automated sequencer (model 373; Applied Biosystems, Inc.), and a set of 12 16-mer primers designed to anneal to sequences 003ف bp apart in the PDR3 gene.

Statistical Analysis
Statistical analysis was performed with the InStat statistics software from GraphPad Inc. (San Diego, CA). 2 analysis and two-tailed P values were obtained from 3 ϫ 2 contingency tables, or the Student's t test was used to compare the means and calculate a two-tailed P value from analysis of variance.

Analysis of M-C 6 -NBD-PE Influx and Efflux
When the diploid strain, CRY3, was incubated at 30ЊC in the presence of donor vesicles containing N-Rh-DOPE, DOPC, and either M-C 6 -NBD-PE or M-C 6 -NBD-PC, the rapidly exchangeable lipids, M-C 6 -NBD-PE and M-C 6 -NBD-PC (33), were internalized (Fig. 1). The absence of rhodamine fluorescence in the cells (not shown) indicated that the nonexchangeable fluorescent lipid, N-Rh-DOPE (35), was not internalized. Thus, the accumulation of intracellular NBD fluorescence was not due to fusion of donor vesicles or internalization of intact vesicles, but rather internalization of the NBD-phospholipid from the outer leaflet of the plasma membrane after transfer from the donor vesicles (26). Fig. 1 shows that M-C 6 -NBD-PC and M-C 6 -NBD-PE are sorted to different organelles in yeast. M-C 6 -NBD-PC was transported primarily to the vacuole (the yeast lysosome) and, to a much smaller extent, to the mitochondria and nuclear envelope (as previously documented, [26]), while M-C 6 -NBD-PE was excluded from the vacuole and instead accumulated in the mitochondria and nuclear envelope, as determined by colocalization with DAPI ( Fig. 2), and perhaps other unidentified organelles. Fig. 3 shows a time course of M-C 6 -NBD-PE internalization and efflux by a combination of fluorescence microscopy, spectrofluorometry, and quantitation after extraction and separation by TLC. The spectrofluorometric internalization assay (Fig. 3 A) measured the increase in NBD-fluorescence as M-C 6 -NBD-PE transferred from the quenching environment of the donor vesicles to the nonquenching environment of the yeast cellular membranes. The accuracy of the spectrofluorometric assay, as well as fluorescence microscopy digital imaging, to measure the accumulation of M-C 6 -NBD-PE were confirmed by concomitant extraction and TLC separation. No degradation products of M-C 6 -NBD-PE were detected in the cell extracts, indicating that only intact M-C 6 -NBD-PE was internalized and that either intracellular degradation was insignificant or that degraded products were rapidly effluxed from the cell. All three techniques illustrated that the net amount of internalized M-C 6 -NBD-PE steadily increased during an hour of incubation with the donor vesicles (Fig.  3, A and D).
After the net internalization of M-C 6 -NBD-PE, cells were washed free of the donor vesicles and incubated at 30ЊC with (Fig. 3, C and F) or without (Fig. 3, B and E) excess unlabeled acceptor vesicles, and the NBD fluorescence was monitored by the three techniques described above. Since NBD fluorescence is highly sensitive to the polarity of its immediate environment (32,34), degradation of M-C 6 -NBD-PE to water-soluble products resulted in almost complete loss of fluorescence. Thus, the data presented in Fig. 3, B and E, illustrated a time-dependent degradation of M-C 6 -NBD-PE. To determine whether the degradation occurred intracellularly or was subsequent to efflux from the cells, the rate of loss of cellular fluorescence and degradation were monitored in the presence of excess unlabeled acceptor vesicles. Removal of the intact probe from the cell surface by its transfer to the acceptor vesicles would be expected to prevent its degradation and therefore inhibit the overall rate of loss of the fluorescence signal. The result (Fig. 3, C and F) illustrated that concomitant with its loss from the cells, intact M-C 6 -NBD-PE appeared in the acceptor vesicle-containing medium. This result was confirmed by the slower rate of degradation detected by fluorometry (Fig. 3 C). These data indicated that intact M-C 6 -NBD-PE was effluxed from the cells and degraded. The existence of an extracellular phospholipase B capable of hydrolyzing NBD-labeled lipids has been described previously (28,56). These data are consistent with the interpretation that in the presence of excess M-C 6 -NBD-PE-containing donor vesicles, the concurrent influx and efflux resulted in net intracellular accumulation. Upon removal of the donor vesicles, the net flux reversed, resulting in degradation of M-C 6 -NBD-PE by extracellular phospholipases unless it was removed from the cell surface by transferring to acceptor vesicles in the medium.
Similar results were obtained when the above experiment was performed with the temperature-sensitive sec6-4 strain. At the nonpermissive temperature (37ЊC), the transport of secretory vesicles to the plasma membrane in this strain is blocked, resulting in the accumulation of ex-cess intracellular secretory vesicles (53). Numerous sec mutants, including sec6-4, that inhibit vesicular traffic (36) also inhibit the endocytic uptake of M-C 6 -NBD-PC (26) and the lipophillic membrane probe, FM4-64 (52). Thus, the observation that M-C 6 -NBD-PE internalization and efflux occurred at rates similar to that observed in the wild-type Sec ϩ strain (Fig. 3, A and C) indicated that M-C 6 -NBD-PE influx was not dependent on the same pathway as M-C 6 -NBD-PC and FM4-64 endocytosis nor was its efflux dependent on secretion.
Additional sec mutant strains were tested for their ability to accumulate M-C 6 -NBD-PE by measuring the ratio of M-C 6 -NBD-PE accumulation (measured by average pixel brightness) at the nonpermissive temperature (37ЊC) to that at the permissive temperature (23ЊC). This ratio for sec1, 7,11,12,14, and 18 ranged from 70 to 97% of their isogenic Sec ϩ strains. Thus, M-C 6 -NBD-PE internalization occurs even when the endocytosis of M-C 6 -NBD-PC and FM4-64 are inhibited. In addition, the observations that M-C 6 -NBD-PE is not transported to the vacuole (Figs. 1 and 2) and is not degraded intracellularly further support the conclusion that M-C 6 -NBD-PE is internalized primarily by a mechanism that does not require endocytosis. M-C 6 -NBD-PE transport in yeast possessed many of the same characteristics as NBD-labeled phosphatidylethanolamine sorting in mammalian cells. In addition to targeting to the same intracellular organelles, M-C 6 -NBD-PE transport in yeast was inhibited by ATP depletion, low temperature, and NEM treatment (Fig. 4). Preincubation of the CRY2 strain with SCNaN 3 medium depleted cellular ATP by 80% (26) and reduced the accumulation of M-C 6 -NBD-PE to 7% of the pixel brightness of the untreated control cells. Incubation at 2ЊC reduced accumulation to 10% of controls. Pretreatment and labeling of CRY2 cells at 2ЊC in the presence of 2 mM NEM further reduced the pixel brightness to 4%. The similarity of these characteristics of M-C 6 -NBD-PE traffic in yeast to NBD-labeled phosphatidylethanolamine traffic in mammalian cells suggests that it is translocated across the yeast plasma membrane by a protein analogous to the mammalian aminophospholipid translocase (31,45,49).

Isolation of Mutants That Do Not Accumulate M-C 6 -NBD-PE
We developed a novel scheme for isolating yeast mutants (referred to as tpe mutants) that were defective in the accumulation of M-C 6 -NBD-PE. As shown above (Figs.  1-3), wild-type yeast cells became highly fluorescent when they internalized M-C 6 -NBD-PE. Exposure of the labeled cell cultures to intense light between 470-480 nm (the excitation maximum for NBD) for 2 h resulted in the death of Ͼ95% of the cells. Photokilling required both incubation with vesicles and illumination. Elimination of either incubation condition eliminated the photokilling. Mu-tagenized cultures were subjected to five "rounds" of photokilling enrichment (described in detail in Materials and Methods) and then plated for single colonies. Individual colonies were examined microscopically for defects in M-C 6 -NBD-PE accumulation. Testing multiple individual colonies that grew on rich medium containing glucose (YPD medium) after five rounds of dye-sensitized photokilling revealed a Ͼ25:1 ratio of tpe mutants/wild-type survivors. 8-10 Tpe Ϫ survivors from each independently mutagenized culture were saved for further analysis. Fig. 5 shows the phenotype of tpe mutants representing two different complementation groups (see analysis below) obtained from the photokilling enrichment. After incubation with donor vesicles containing M-C 6 -NBD-PE, tpe mutants showed no fluorescence when microscopy was performed with a neutral density filter that allowed 5% of the light from a 100-W lamp to reach the sample, although in the Tpe ϩ parent strain, highly fluorescent mitochondria and nuclear envelopes were apparent (Fig. 5, compare A  and D). When the neutral density filter was removed, low levels of intracellular and cell surface fluorescence could be observed in both tpe mutants, whereas fluorescent emission from the Tpe ϩ cells was oversaturated (Fig. 5,  compare B, E, and H). Inhibition of the M-C 6 -NBD-PE accumulation into the tpe mutants detected by fluorescence microscopy was confirmed by extraction of total cellular lipids and recording of total NBD fluorescence in the extract as well as the fluorescence of M-C 6 -NBD-PE purified from the extract by TLC. Thus, the lack of fluorescence detected by microscopy reflected a quantitative decrease in M-C 6 -NBD-PE cellular accumulation, and the "ring staining" pattern in the tpe mutants presumably re-

Genetic Analysis of the tpe Mutants
The Tpe Ϫ clones were placed into two complementation groups by partial complementation of the M-C 6 -NBD-PE accumulation defect. Two independent isolates from the first complementation group and one from the second were selected for further characterization. These three mutant strains were back-crossed three times against the Tpe ϩ parent strains to produce congenic Tpe Ϫ strains. Tpe ϩ /Tpe Ϫ phenotypes segregated 2:2 in each of the nine tetrads analyzed per back-cross for all three Tpe Ϫ strains, indicating that for each tpe allele, a single gene was re- Figure 3. Cellular M-C 6 -NBD-PE influx and efflux measured by fluorescence microscopy, fluorometry, and quantitative TLC. CRY2 was grown to mid-log phase in SDC medium at 30ЊC. NY17 (sec6-4) was grown to mid-log in SDC at room temperature (32فЊC). The sec6-4 cells were warmed to 37ЊC for 30 min before labeling with M-C 6 -NBD-PE and were treated identically to the CRY2 cells as described below with the exception that they were maintained at 37ЊC during influx and efflux instead of 30ЊC. CRY2 cells were pelleted and resuspended in SDC ϩ 2% sorbitol at OD 600 ϭ 0.16. Labeling of cells was initiated by the addition of M-C 6 -NBD-PE-containing donor vesicles (vesicle phospholipid concentration, 50 M). A 2-ml aliquot was immediately placed in a stirred fluorometer cuvette at 30ЊC and NBD fluorescence (excitation, 475 nm; emission, 530 nm) was recorded continuously. The remainder were placed in a shaker incubator at 30ЊC. Aliquots were removed at the indicated times and washed three times in SCNaN 3 . A small aliquot was removed for fluorescence microscopy and the remainder were extracted, separated by TLC, and quantified by digital imaging of the M-C 6 -NBD-PE fluorescent spots. After 1 h, the remaining labeled cells were washed three times in ice-cold SDC ϩ 2% sorbitol. Measurement of M-C 6 -NBD-PE in the cells and medium post labeling was initiated by returning the cells to 30ЊC in the shaker incubator in the presence or absence of unlabeled DOPC vesicles (50 M). 2-ml aliquots were removed and placed in the fluorometer for continuous recording as above. Aliquots were removed at the indicated times for fluorescence microscopy, TLC separation and quantification as described sponsible for the Tpe Ϫ phenotype. Diploids containing one Tpe ϩ and one Tpe Ϫ allele were not capable of accumulating M-C 6 -NBD-PE to wild-type levels. Diploids resulting from crosses of the two isolates from the first complementation group to their isogenic parent strain remained defective in M-C 6 -NBD-PE accumulation, and thus, these two mutant alleles appeared to be dominant. The isolate from the second complementation group when crossed to its parent strain showed a partial defect in M-C 6 -NBD-PE accumulation and was considered to be semidominant. The two dominant Tpe Ϫ strains were assigned to the TPE1-1 and TPE1-2 (capitals to denote dominance) complementation group and the semidominant strain was assigned to tpe2-1.

tpe Mutants Are Temperature-sensitive for Oxidative Phosphorylation
Growth of TPE1-1 and tpe2-1 mutants was temperaturesensitive on nonfermentable carbon sources (Fig. 6), indicating a temperature-sensitive loss of respiratory function in the mutants. After the third back-cross of TPE1-1, TPE1-2, and tpe2-1, the segregation pattern of the respira- Figure 4. M-C 6 -NBD-PE transport in yeast is ATP-, temperature-and NEM-sensitive. Cells were treated with SCNaN 3 medium (to deplete ATP stores), low temperature, or NEM as described in Materials and Methods. M-C 6 -NBD-PE internalization assays, fluorescence microscopy, and pixel brightness analysis were then performed as described in Materials and Methods. At least 20 cells were analyzed to determine the mean pixel brightness for each condition. These numeric values are shown above each column. Figure 5. tpe mutants are inhibited in the accumulation of M-C 6 -NBD-PE. TPE1-1 and its Tpe ϩ parent strain, CRY2, were grown to midlog phase at 23ЊC. The incubation temperature was then shifted to 37ЊC for 30 min, and M-C 6 -NBD-PE internalization assays were performed for 1 h before microscopic analysis as described in Materials and Methods. (C, F, and I) DIC optics. In A and D, a neutral density filter was used that attenuated the excitatory light by 95%. In B and E, the filter was removed to allow 100% of the excitatory light to impinge upon the sample. tory deficiency phenotype (Respiratory Ϫ ) was determined. In each of the nine tetrads dissected for each mutant allele, Respiratory ϩ /Respiratory Ϫ segregated 2:2, indicating that a single gene was responsible for this phenotype. Furthermore, the Respiratory Ϫ and M-C 6 -NBD-PE-accumulation defect cosegregated in 100% of the tetrads analyzed, indicating that the same gene or a tightly linked gene was responsible for both phenotypes. tpe2-1 exhibited a more severe respiration defect than the TPE1-1 mutant, being unable to grow on glycerol/ethanol medium (YEP-GE medium) (Fig. 6) or lactate (not shown) at 30ЊC, whereas the TPE1-1 mutant exhibited this growth defect only at 37ЊC. Both TPE1-1 and tpe2-1 mutants grew on YEP-GE medium at 23ЊC and on the fermentable carbon sources glucose (YPD medium) (Fig. 6) and galactose (YP-Gal medium) (not shown) at 23, 30, and 37ЊC. TPE1-1/tpe2-1 double mutants exhibited synthetic lethality in that growth on YPD at 37ЊC and growth on YP-glycerol at 23ЊC was inhibited (not shown).
Although the tpe mutants did not arrest when grown in fermentable carbon sources at 37ЊC, they did exhibit growth defects under these conditions. Growth of TPE1-1 and tpe2-1 in YP-Gal was slowed twofold compared to the isogenic Tpe ϩ strains, and growth on glucose was reduced by 1.6-fold in rich media (YPD) and fivefold in synthetic media (SDC). Thus, although the growth defects at 37ЊC in the tpe mutants were predominantly manifested on nonfermentable carbon sources, to some extent growth defects were observed on all media tested.

The Percentage of Endogenous Phosphatidylethanolamine Exposed to the Outer Leaflet of the Plasma Membrane Is Increased in the tpe Mutants
To determine whether the transport of M-C 6 -NBD-PE across the plasma membrane was indicative of the behavior of endogenous phosphatidylethanolamine, the amount of phosphatidylethanolamine in the outer leaflets of TPE1-1, tpe2-1, and their parent Tpe ϩ strain was measured by labeling with TNBS using membrane impermeant conditions. The percentage of TNP-labeled phosphatidylethanolamine increased four to fivefold in both mutants relative to the Tpe ϩ strain (Table II). Thus, the amount of phosphatidylethanolamine in the outer leaflet of the plasma membrane increased in the mutant strains consistent with a decrease in the net influx rate of endogenous phosphatidylethanolamine as well as M-C 6 -NBD-PE.

tpe Mutants Are Defective in Membrane-Lipid Endocytosis
The effect of the tpe mutations on endocytosis was tested for two different membrane markers. M-C 6 -NBD-PC (26) and FM4-64 (52) have been shown previously to be endocytosed to the vacuole in S. cerevisiae. As shown in Table III, rows B and C, accumulation of both probes was inhibited to similar extents in the tpe mutants relative to the parent strain. Although the extent of accumulation of both probes was attenuated, FM4-64 and M-C 6 -NBD-PC fluorescence was localized to the vacuolar membrane and lumen, respectively (data not shown). Thus, there was a generalized defect in endocytosis of membrane-lipids in the tpe mutants. It is important to note that although endocytosis was inhibited in the tpe mutants, it was not reduced as severely as was the nonendocytic traffic of M-C 6 -NBD-PE.   TPE1-1, and tpe2-1, respectively. Data are reported as the mean and standard error of four independent analyses from one experiment that was representative of three.
Estimates of the extent of M-C 6 -NBD-PC uptake compared to M-C 6 -NBD-PE by pixel brightness analysis showed that there was at least a 10-fold greater accumulation of M-C 6 -NBD-PC than M-C 6 -NBD-PE in the tpe mutants at 37ЊC (Table III, rows A and B).
The nonspecific internalization in the TPE1-1 and tpe2-1 strains of other amphipathic fluorescent probes, including DAPI and 3,3Ј-dihexyloxacarbocyanine iodide (DiOC 6 ) (data not shown), to the same level as their isogenic parent strains indicated that inhibition of NBD-lipid accumulation was not due to a nonspecific block of internalization of all lipophillic molecules.

tpe Mutants are Drug Resistant
Given the identification of MDR1 and MDR2 as phospholipid transporters (40,46,51), we tested the possibility that the sorting defects in the tpe mutants were due to the upregulation of an MDR-like efflux pump. Activation of a variety of MDR homologues in yeast leads to increased resistance to a large array of drugs because of enhanced efflux of these drugs from the cells (5,7). If the tpe phenotype resulted from an activated MDR-like pump, mutants would also be expected to display increased drug-resistant phenotypes. We therefore tested the sensitivity of TPE1-1, tpe2-1, and their parent Tpe ϩ strains to three structurally unrelated drugs, oligomycin, 4-nitroquinoline N-oxide (4-NQO), and cycloheximide, using a spot test assay (see Materials and Methods). Resistance to cycloheximide and 4-NQO has been shown to result primarily from the amplification of the ABC transporters, Pdr5p and Snq2p, re-spectively (12,24,29). Resistance to oligomycin is at least partly due to the amplification of Yor1p (25). Table IV shows that both TPE1-1 and tpe2-1 are resistant to 4-NQO and cycloheximide at 30ЊC and are resistant to oligomycin at 23ЊC, compared to the isogenic Tpe ϩ strain. The isogenic Tpe ϩ strain does not grow on YPD plates containing 0.2 g/ml cycloheximide or greater; however, both tpe mutants grow on plates containing 1 g/ml cycloheximide (Table IV). TPE1-1 shows a slightly greater resistance to oligomycin and 4-NQO than tpe2-1; however, they both are significantly more resistant than the Tpe ϩ strain (Table IV). The relative resistance of TPE1-1 and tpe2-1 to cycloheximide and 4-NQO was similar at 23, 30, and 37ЊC. Tests of oligomycin resistance required growth on a nonfermentable carbon source. Therefore, because of the temperature-sensitive growth defect of the mutants, tests were limited to 23ЊC.

Identification of TPE2 and TPE1
Complementation of the temperature-sensitive growth defect on nonfermentable carbon sources of tpe2-1 was used to isolate the TPE2 gene (see Materials and Methods). Transformation with PDR3 resulted in full complementation of the M-C 6 -NBD-PE accumulation defect (Fig. 7 A), cycloheximide resistance (Fig. 7 B), and the temperaturesensitive growth defect on nonfermentable carbon sources of tpe2-1 (data not shown). Linkage analysis determined that tpe2-1 was allelic with PDR3 (38). PDR3 has been identified as a transcriptional activator of the pleiotropic drug resistance network (14).
We hypothesized that TPE1 was identical to PDR1 and therefore tested the linkage of these two genes based on the following three observations: (a) TPE2 and PDR3 were determined to be identical; (b) PDR1 is a transcriptional activator of the drug resistance network and is homologous to PDR3 (5); and (c) TPE1-1 is drug resistant (Table IV). Tetrad analysis of TPE1-1 and PDR1-3 for cycloheximide resistance indicated that TPE1 and PDR1 were tightly linked.

TPE1-1 and tpe2-1 Are Respective Gain-of-Function Alleles of PDR1 and PDR3
To test the predicted identity of TPE1 with PDR1 and TPE2 with PDR3, the nonessential genes PDR1 and PDR3 were deleted in both tpe mutant strains. Deletion of Yeast strains were grown to mid-log phase, and lipid internalization assays, microscopy, and pixel brightness analysis were perfomed as described in Materials and Methods. For each determination, the mean pixel brightness of 10-38 cells and standard deviation were determined. For each internalization condition, the pixel brightness of CRY2 was set to 100%, and the pixel brightness in the other strains is shown as a percentage of this value. The pixel brightness of CRY2 under the various incubation conditions was: (A) 177 Ϯ 28; (B) 132 Ϯ 50; (C) 12 Ϯ 7.
PDR1 in the TPE1-1 mutant strain restored wild-type levels of M-C 6 -NBD-PE accumulation (Fig. 8) and cycloheximide sensitivity (data not shown). Similarly, PDR3 deletion in the tpe2-1 strain also restored wild-type levels of M-C 6 -NBD-PE accumulation and cycloheximide sensitivity. Conversely, deletion of PDR1 in tpe2-1 and deletion of PDR3 in TPE1-1 had no effect on either mutant phenotype. These data indicated that TPE1-1 is a gain-of-function mutant allele of PDR1, and tpe2-1 is a gain-of-function mutant allele of PDR3. Consistent with this interpretation, deletion of PDR1 and PDR3 in the parental Tpe ϩ strain resulted in wild-type levels of M-C 6 -NBD-PE accumulation and cycloheximide sensitivity (data not shown). From this point on, TPE1-1 will be referred to as PDR1-11, and tpe2-1 will be referred to as pdr3-11.

The Defect in M-C 6 -NBD-PE Accumulation in PDR3 Is Allele Specific
The strain carrying the mutant allele pdr3-2 was originally isolated based on its resistance to chloramphenicol and cycloheximide (20) and subsequently identified as an allele of PDR3 (48). This strain was compared to the tpe2-1/ pdr3-11 allele in relation to M-C 6 -NBD-PE accumulation and drug resistance (Fig. 9, Table IV). Allele specificities were observed in both. Unlike tpe2-1/pdr3-11, pdr3-2 did not exhibit a defect in M-C 6 -NBD-PE accumulation; however, both alleles exhibited similar resistance to cycloheximide (Table IV). pdr3-2 was also less resistant to 4-NQO and oligomycin than tpe2-1.
The PDR3 mutant allele, tpe2-1/pdr3-11, was rescued by integrative transformation followed by plasmid eviction of the mutant allele. Sequencing revealed a single point mutation. Glycine 957 , which is predicted to be in the activation domain, was replaced by an aspartate.

Model for M-C 6 -NBD-PE Translocation
The data presented here provide evidence that M-C 6 -NBD-PE is internalized into yeast by a mechanism that is distinct from the major pathway for M-C 6 -NBD-PC internalization. This mechanism is detailed in the model shown in Fig. 10. In step 1, M-C 6 -NBD-PE molecules dissociate from donor vesicles and insert in the outer leaflet of the yeast plasma membrane. Even though M-C 6 -NBD-PE molecules are predominantly membrane bound (their molar concentration in the membrane is 10 7 times greater than their concentration in water [33]), they rapidly exchange through the medium between membranes (t 1/2 ϭ 2ف s at 25ЊC [33]). Furthermore, by incorporating a "non-  pase model, in which an amphiphile partitions into one leaflet of a membrane and its polar group is flipped to the other side (21), is the most likely mechanism to explain M-C 6 -NBD-PE translocation across the plasma membrane. These characteristics of M-C 6 -NBD-PE internalization in yeast are similar to the characteristics of the flippase-mediated phosphatidylethanolamine uptake that has been described in mammalian cells (10,31,42) and yeast (3).
Our results show that the predominant pathway for M-C 6 -NBD-PE internalization occurs even when lipid endocytosis is inhibited. M-C 6 -NBD-PE is not delivered to the vacuole like other lipid markers that are internalized by endocytosis, nor is its internalization inhibited in sec mutants shown previously to inhibit lipid-endocytosis (26,52). However, although M-C 6 -NBD-PE accumulation occurs when endocytosis is inhibited, our results do not exclude the possibility that under normal conditions, topological translocation of M-C 6 -NBD-PE to the cytoplasmic surface also occurs subsequently to the early steps of endocytosis.
After translocation to the cytoplasmic leaflet of the plasma membrane or endocytic vesicle, M-C 6 -NBD-PE is transported to intracellular organelles including, but not necessarily limited to, the nuclear envelope and mitochondria (step 4). Given the rapid rate of exchange of M-C 6 -NBD-PE between membranes measured in vitro (33), it is likely that transfer from the inner leaflet of the plasma membrane to other intracellular organelles occurs spontaneously. However, the different organelle membranes are not labeled to the same extent. For example, M-C 6 -NBD-PE labeling was clearly seen in the mitochondria and nuclear envelope but never in the vacuolar membrane. Thus, the rapidly exchanging M-C 6 -NBD-PE molecules appeared to be sorted into distinct organelle membranes. The observed sorting may result from directed transport to specific organelles or it may reflect an equilibrium distribution determined by the properties of the organelle membranes. At this time, the mechanisms responsible for the apparent nonuniform labeling of the intracellular membranes is unknown.
In addition to its endocytosis-independent internalization, our results also provide evidence (Fig. 3) for the efflux of intact M-C 6 -NBD-PE molecules from yeast cells (step 3). It is important to realize that although we monitor this efflux only after the cells are loaded with M-C 6 -NBD-PE, influx and efflux are likely occurring concurrently. In the presence of medium containing excess M-C 6 -NBD-PE donor vesicles (Fig. 3, A and D), the balance leads to intracellular accumulation of fluorescent lipid. When these donor vesicles are removed, the balance shifts to favor a depletion of intracellular fluorescence. Our data do not distinguish whether a single protein is responsible for both influx and efflux, or whether influx and efflux are mediated in different directions by different proteins.

Relevance of Short Acyl Chain, NBD-labeled Phospholipid Trafficking to Endogenous Phospholipid Trafficking
Although NBD-labeled lipids have been used for many years to study lipid sorting and trafficking in cultured cells (37), one must consider the extent to which the presence of the NBD group and/or the truncation of the sn-2 acyl chain alters the behavior of the phospholipid analogue relative to its endogenous counterpart. The differential pattern of accumulation of M-C 6 -NBD-PE relative to M-C 6 -NBD-PC (Fig. 1) demonstrated unequivocally that the yeast cells distinguish between the head group structures of these two molecules and import them to different locations. Since the diacyl glycerol portion of these two molecules is identical, one can conclude that the NBD group does not determine the uptake pathway nor does it interfere with the discrimination of different head groups by the yeast plasma membrane.
It is clear that truncation of one or both acyl chains of a phospholipid molecule has a profound effect on its stability in membranes. Based on the relative rates of transfer through the water phase between liposomes, 1-palmitoyl, 2-oleoyl phosphatidylcholine is 1.4 ϫ 10 5 times (23) and dimyristoyl phosphatidylcholine is 3,000 times (23) less likely to dissociate from a membrane into the water than M-C 6 -NBD-PC (33). Thus, the usefulness of truncated NBD-labeled lipids for studying the effect of acyl chain length and structure on intermembrane transfer is limited. However, as stated above, even though the truncated NBD-labeled lipids are significantly more water soluble, their concentration in membranes is still 10 7 times greater than in water (33). Thus, even though they are readily exchangeable, they exist predominantly in the membrane.

PDR1 and PDR3 Regulate M-C 6 -NBD-PE and Endogenous Phosphatidylethanolamine Net Translocation Across the Plasma Membrane
A novel mutant enrichment procedure based on photokilling of cells that accumulated normal amounts of M-C 6 -NBD-PE was developed to identify genes required for the accumulation of intracellular M-C 6 -NBD-PE. Two genes, originally named TPE1 and TPE2, were identified and found to be identical to two previously identified pleiotropic drug resistance genes, PDR1 (6) and PDR3 (14), respectively. PDR1 and PDR3 are transcription regulators that control the transcription of numerous genes involved in a wide range of functions, encompassing resistance to chemical and physical stresses, membrane transport, and organelle function (5). Gain-of-function alleles of PDR3 mediate resistance to a wide variety of drugs by activating transcription of the corresponding ABC transporters that efflux the drugs out of the cells. ABC transporters shown to be regulated by PDR3 include YOR1 (25), SNQ2 (12,29), and PDR5 (8,24), which mediate the efflux of oligomycin, 4-NQO, and cycloheximide, respectively. PDR3 autoregulates itself (13) and mediates resistance to other drugs as well and is consequently believed to regulate other genes (4,5). PDR3 is most homologous (36% identical) and functionally analogous to another transcriptional activator of this PDR family, PDR1, which also mediates drug resistance through the aforementioned ABC transporters. Preliminary evidence suggests that PDR1 may be involved in the regulation of a host of other genes such as PDR10, PDR15, GAS1, glycerol-3-phosphate dehydrogenase (G3PD), and others as well as PDR3.
The observation that M-C 6 -NBD-PE appears to flipflop in both the inward and outward directions suggests that the steady-state amount of M-C 6 -NBD-PE accumulation resulting from our standard assay is determined by the relative rates of each process. In wild-type cells, M-C 6 -NBD-PE is accumulated because the rate of inwarddirected flippase activity exceeds the outward-directed activity. Given that drug resistance is conferred by increasing the rate of drug efflux from yeast cells and the demonstration that a mechanism for M-C 6 -NBD-PE efflux exists in wild-type cells (Fig. 3), it is likely that TPE1-1/PDR1-11 and tpe2-1/pdr3-11 activate the transcription of one or more flippases that actively transport M-C 6 -NBD-PE in the outward direction. However, one cannot rule out the possibility that these mutant alleles decrease the activity of an inward-directed flippase either by inhibiting its transcription or by activating the transcription of an inhibitor.
In addition to a decreased net inward flux of M-C 6 -NBD-PE, TPE1-1/PDR1-11 and tpe2-1/pdr3-11 strains also exhibited a four to fivefold increase in the amount of endogenous phosphatidylethanolamine exposed in the outer leaflet of the plasma membrane (Table II). This result supports the assertion that the observed changes in M-C 6 -NBD-PE accumulation in the tpe mutants reflect alterations in the distribution of endogenous phosphatidylethanolamine.

Identification of Flippases in Yeast
Several recent reports have identified proteins with flippase activity in mammalian cells. The ABC transporters, human MDR1 and MDR3, as well as mouse mdr2, have been shown to have outward-directed phospholipid flippase activity (40,47,51). This suggests the likely possibility that the flippase responsible for the outward-directed flipflop of M-C 6 -NBD-PE will be included within a limited class of yeast homologues of ABC transporters that contain the consensus sequence for the DNA binding domain of PDR1 in their promoter regions (5). Inward-directed phosphatidylserine flippase activity has been reported to be encoded by the DRS2 gene-a yeast homologue of a bovine chromaffin granule, P-type ATPase. Deletion of DRS2 results in the inability to internalize NBD-labeled phosphatidylserine across the plasma membrane (49). Using our standard labeling protocol, a DRS2 deletion strain accumulated M-C 6 -NBD-PE to the same extent as its isogenic Drs2 ϩ parent strain (Grant, A.M., and J.W. Nichols, unpublished observation). Thus, DRS2 is not solely responsible for the internalization of M-C 6 -NBD-PE. Several genetic approaches are available that should allow the identification of the inward-and outwarddirected flippases and the mechanisms involved in the regulation of phosphatidylethanolamine transport and its steady-state distribution across the plasma membrane.

Significance of Flippases in Unicellular Organisms
Numerous mammalian cell types (for reviews see references 15,16,54), as well as the yeast Saccharomyces carlbergensis (9), have been reported to have an asymmetric distribution of phospholipids across their plasma membranes. In general, the aminophospholipids, phosphatidylserine and phosphatidylethanolamine, are sequestered to the inner leaflet while the choline lipids, phosphatidyl-choline and sphingomyelin, and glycolipids are concentrated in the outer leaflet. Establishment of this asymmetric distribution is thought to result from the activity of one or more aminophospholipid translocases that flip-flop phosphatidylserine and phosphatidylethanolamine from the outer to inner leaflet. Although the loss of the asymmetric distribution of phosphatidylserine acts as a signal for several intercellular or systemic signaling events, including blood coagulation (39), clearance of senescent red cells (11), and phagocytosis of apoptotic cells (17,18), the role of the establishment and maintenance of an asymmetric phospholipid distribution for the function of single cells has yet to be determined. It has been proposed that inward-directed phosphatidylserine and/or phosphatidylethanolamine translocation is required for the inward budding of the plasma membrane during endocytosis (15). Active inward translocation of these lipids is hypothesized to increase the phospholipid surface density in the inner leaflet, inducing inward bending of the bilayer as predicted by the bilayer-couple hypothesis (43). The observation that mutant strains defective in M-C 6 -NBD-PE accumulation (PDR1-11 and pdr3-11) are also defective in the endocytosis of lipid probes is consistent with this hypothesis. Regardless of whether M-C 6 -NBD-PE accumulation in the mutants is inhibited by an increased efflux or a decreased influx, the net result would be a loss of the ability to establish the excess surface density in the inner leaflet predicted to be required for inward vesicle budding.
The data presented here establish that yeast cells have the ability to translocate M-C 6 -NBD-PE in the inward and outward direction and that the net direction of translocation is under transcriptional regulation by PDR1 and PDR3. This suggests a fundamental cellular role for phospholipid flippases unrelated to intercellular signaling and interactions. The presence of flippases that pump in opposite directions suggests that yeast cells have the ability to coordinately regulate their activities to produce dynamic membrane perturbations or to produce an appropriate transbilayer phospholipid distribution for their survival in a wide range of environments during different stages of growth.


extfieldsofstudy: Biology Medicine

text:
Induction of DNA synthesis and apoptosis are separable functions of E2F-1.

The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E2F-1, suggesting that it may be mediated through alleviation of E2F-dependent transcriptional repression. These results indicate that E2F-1 can show independent cell cycle progression and apoptotic functions, consistent with its putative role as a tumor suppressor.

Cell cycle progression is a tightly regulated process whose fidelity and ordered progression is monitored at several control points throughout the cycle. Tumor cells frequently show loss of the normal function of these checkpoints, which can lead to unrestrained proliferation and the acquisition of genetic instability (Hartwell and Kastan 1994). Several of the best understood tumor suppressor proteins such as the retinoblastoma gene product pRB, p53, and p16, whose function is frequently lost in many human cancers, play critical roles in maintaining normal cell growth by exerting negative controls on cell cycle progression (Sherr 1996). Although these proteins have multiple functions, one common feature is their ability to target the activity of the E2F family of transcription factors, one of the key mediators of cell cycle progression into S phase (LaThangue 1994). The importance of E2F function has been demonstrated by studies showing that inhibition of E2F prevents entry into S-phase (Wu et al. 1996) and that ectopic expression of E2F in quiescent cells is sufficient for progression into DNA synthesis (Johnson et al. 1993). Active E2F consists of a heterodimer between one of the E2F proteins (E2F1-5) and a DP-binding partner (DP1-3) (Wu et al. 1995). Regulation of E2F transcriptional function is controlled at several levels, including expression of the E2F compo-~Corresponding author. E-MAIL vousden@ncifcrf.gov~ FAX (301) 846-1666. nents (Kaelin Jr. et al. 1992;Slansky et al. 1993), phosphorylation of each binding partner (Krek et al. 1994) and interaction with one of the pRB family of pocket proteins that includes pRB, p107, and p130 (Weinberg 1995). Although this interaction does not inhibit the DNA-binding activity of E2F, when complexed with pRB, E2F is converted from an activator of transcription to a potent transcriptional repressor (Adams and Kaelin 1995). Indeed, although some of the genes necessary for cell cycle progression are transcriptionally activated by E2F, several genes have been shown to be actively transcriptionally repressed through E2F-binding sites within their promoters, with activation of expression being related to loss of repression, rather than positive transcriptional acti~Tation (Lam and Waston 1993;Johnson et al. 1994;Neuman et al 1994). During cell cycle progression, transcriptionally repressive complexes of E2F with pRB seen during G1 are lost as a result of phosphorylation of pRB by a member of the G1 cyclin dependent kinases, which leads to the dissociation of the pRB/E2F complex (Weinberg 1995). Loss of transcriptional repression and the generation of free E2F with positive transcriptional activity results in the expression of genes necessary for DNA synthesis and progression into S phase. Later, at the end of S phase, cyclin A-associated kinase directly complexes with the E2F component of the transcription factor that is subsequently inactivated following phosphorylation of the DP subunit (Krek et al. 1995). This inactivation of E2F function allows exit from S-phase and continued cell cycle progression.
Despite the clear importance in allowing cell cycle progression, recent studies have shown that one of the components of the E2F family, E2F-1, also shows apoptotic functions under conditions of deregulated expression. In mice, interference with the regulation of E2F activity provided by the pRB protein family, either by deletion of the pRB gene or expression of viral proteins that inhibit pRB function, results in unregulated cell proliferation and apoptosis (Pan and Griep 1994;Almasan et al. 1995). Similarly, ectopic expression of E2F-1 in quiescent fibroblasts leads to entry into DNA synthesis and apoptosis (Qin et al. 1994;Shan and Lee 1994;Kowalik et al. 1995). Several proteins that play a role in regulating cell cycle progression also have apoptotic activities and it has been suggested that the apoptotic signal induced by such proteins is a direct consequence of their growth control functions. In such a conflict of signals model, simultaneous expression of growth promoting and growth arrest signals results in the activation of the cell death pathway. Cooperation between E2F activity and the tumor suppressor protein p53 (Morgenbesser et al. 1994;Wu and Levine 1994) has lead to the suggestion that cell death may be mediated by a conflict of growth stimulatory signals activated by E2F and the growth inhibitory functions of p53. This model implies that the apoptotic activity of deregulated E2F is a consequence of unscheduled cell cycle progression induced by E2F itself and is supported by data from several studies. In particular, analysis of E2F-l-induced apoptosis in mouse fibroblasts has clearly shown that loss of the normal regulation of E2F-1 function through inactivation by cyclin A at the end of S phase provides an apoptotic stimulus (Krek et al. 1995). The observation that E2F-l-deficient mice show an impaired apoptotic function and increased incidence of cancer development (Field et al. 1996;Yamasaki et al. 1996), however, suggests that E2F-1 may also play a more direct role as a tumor suppressor, independent of E2F induced cell proliferation. It is also of interest to note that loss of E2F-1 function had no profound effect on the development of these mice, illustrating that the cell cycle progression function of E2F-1 is not essential during development.
Although p53 can augment E2F-1-induced apoptosis in several systems, previous studies had suggested that the growth inhibitory effects of E2F-1 are not entirely dependent on p53 (Melillo et al. 1994;Qin et al. 1994Qin et al. , 1995Berry et al. 1996) and pRB has been shown to protect p53 null human cells from apoptosis in an E2F-1 bindingdependent manner (Haas-Kogan et al. 1995). A p53-independent apoptotic activity of E2F is strongly supported by a recent study in mice, showing that deletion of Rb (and therefore deregulation of E2F) results in apoptosis in both p53 positive and negative embryos (Macleod et al. 1996). In this study we have shown directly that E2F-1 has p53-independent apoptotic activities in human cells and that E2F-1 apoptotic function is not dependent on transcriptional activation or the ability to induce cell cycle progression.

Contribution of p53 to E2F-l-induced apoptosis
E2F-1 and p53 have been shown to cooperate in the induction of apoptosis. Expression of p53 leads to both a cell cycle arrest and apoptosis in p53 null human tumor cell lines such as Saos-2 or H1299, although the apoptotic activity of p53 itself can be dissociated from cell cycle arrest (Rowan et al. 1996). To examine the contribution of p53 induced cell cycle arrest to E2F-1-mediated apoptosis, we analyzed Saos-2 cells transfected with E2F-1 and p53 (Fig. 1A) using a flow cytometric assay that measures apoptotic rate at the time of harvesting rather than cumulative apoptosis (Rowan et al. 1996). Although high levels of p53 expression can induce substantial apoptosis in these cells, we chose to examine the effects of lower levels of p53 with a plasmid in which p53 expression is under the control of the MoLTR, a relatively weak promoter in these cells. Under these conditions, p53 induces only a low rate of apoptosis, allowing the retention of sufficient sensitivity to detect any increase in apototic rate. As described in several other systems, an enhanced apoptotic rate was seen following coexpression of E2F-1 and p53 (Fig. 1A). Protein analysis by Western blotting showed expression of the transfected p53 and E2F-1 in these cells (Fig. 1B). Very low levels of endogenous E2F-1 were also seen in the absence of transfected protein, although these endogenous levels were not always within the limits of detection in subsequent analyses.
In addition to enhancing the p53-dependent apoptosis, expression of E2F-1 alone, in the absence of p53, also showed an apoptotic response (Fig. 1A). A closer analysis of cells at 24 and 72 hr post-transfection revealed clearly elevated apoptotic rates in E2F-1 expressing cells, despite the absence of p53 (Fig. 1C), which were significantly enhanced by the absence of serum. The ability of E2F-1 to induce p53-independent apoptosis was confirmed in a second p53 null human tumor line, H1299 (data not shown). To further confirm that E2F-1 expression can lead to apoptosis in the absence of p53, we analyzed transfected Saos-2 cells by staining directly for E2F-1 expression (Fig. 1D). Cells sorted on the basis of E2F-1 overexpression again showed an enhanced apoptotic rate compared with the background, untransfected population. We were concerned that the p53-independent apoptotic function of E2F-1 might be a consequence of excessive overexpression of the exogenous protein, both on the basis of the Western blot analysis (Fig. 1B) and the flow cytometric analysis of transfected cells stained directly with an anti-E2F-1 antibody (Fig. 1D), which revealed cells with a range of fluorescence up to 3 logs higher than background. However, gating out very high expressing cells and analyzing the cell cycle profile of the population of cells with fluorescence <10-fold higher than background generated essentially identical data to those described above (data not shown), indicating that p53-independent E2F-l-induced apoptosis is not dependent on extremely high levels of overexpression. Because our results indicated that E2F-1 has apoptotic activity in Figure 1. E2F-1 induces p53 independent apoptosis in Saos-2 cells. Cells were transiently transfected with 1 lag of p53 and/or E2F-1 expression plasmids as indicated and analyzed by FACS. The increase in apoptosis (as determined by cells with a sub-G1 DNA content) above that is seen in the untransfected population (typically 2%-6% with serum, 5%-15% without serum)is shown. the absence of p53, we continued to analyze this function in p53 null cell lines.

Activation of cell cycle progression is not necessary for E2F-1 induced apoptosis
Although p53 is clearly not necessary for the E2F-l-in-duced apoptotic signal, the well-established cooperation between p53 and E2F-1 may reflect the conflict between E2F-1 proliferative and p53 cell cycle arrest activities, it is also possible, therefore, that elevated apoptosis seen following ectopic expression of E2F-1 in serum deprived Saos-2 cells is the result of the conflicting signals generated by the E2F proliferative signal in the absence of Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from exogenous growth factors. In an attempt to understand the mechanism of E2F-l-mediated p53-independent apoptosis, we utilized a number of previously characterized E2F-1 mutants (Helin and Harlow 1994) (Fig. 2A). Because the ability of E2F-1 to drive cells through cycle has been shown to require an intact DNA-binding and transactivation domain, we sought to analyze the importance of E2F-1 transactivation activity to the apoptotic function. To do this, we tested two E2F-1 mutants in which the transactivation domain was deleted [E2F-l(1-374) and E2F-l(1-284)] and one point mutant [E2F-1(132E)] that fails to bind DNA. As shown in other cell systems (Lukas et al. 1996), all the mutants were transcriptionally inactive as measured by activation of an E2F dependent promoter and all showed dominant-negative ability to inhibit transcriptional activation by the wild-type protein ( Fig. 2B and data not shown). The E2F-1(1-374) and E2F-l(1-284) mutants, which retain the ability to bind DNA, are likely to inhibit wild-type E2F-1 activity by competitively binding to E2F sites. The non-DNA-binding mutant E2F-1(132E) retains the ability to interact with the DP-binding partner and competes with wild-type E2F-1 for the formation of an active dimeric complex. Western blot analysis of cells transfected with 1 ~lg of each E2F-1 expression plasmid showed similar levels of each E2F-1 protein in the transfected cells during the course of these transient assays. However, the two carboxy-terminal deletion mutants E2F-1 (1-374) and E2F-1 (1-284) were consistently expressed at slightly higher levels than the wild-type E2F-1 or E2F-l(E132) proteins. These observations are consistent with the recent observation that sequences within the carboxyl terminus of E2F-1 confer protein instability, and deletion of this region stabilizes the protein (Hoffmann et al. 1996).
To analyze the correlation between promotion of entry into DNA synthesis and apoptosis induced by E2F-1, cell cycle profiles of Saos-2 cells transiently transfected with each E2F-1 mutant were examined in the absence of serum, conditions under which maximum apoptosis was induced by wild-type E2F-1 (Fig. 1C). Although these cells do not become quiescent in the absence of serum, a retardation of cell cycle progression can be seen with Activation of an E2F dependent reporter gene by wild-type E2F-1, but not mutant E2F-1, and repression of wild-type E2F-1 transcriptional activity by E2F-1(1-374) in Saos-2 cells harvested 24 hr post-transfection. (C) Expression of E2F-1 and mutants in Saos-2 cells transiently transfected with 1 jag of each E2F-1 expression plasmid as in B. some accumulation of cells in G1. As shown previously, whereas expression of wild-type E2F-1 accelerates the rate at which cells enter S phase, each of the transcriptionally inactive mutants further slowed progression through the cell cycle leading to an increased accumulation of cells in G 1 (Lukas et al. 1996) (Fig. 3A). This impediment to cell cycle progression is likely to be a consequence of interference with the endogenous E2F function in these cells, either by competing for DNAbinding sites or by titration of the DP-binding partners. Surprisingly, however, both transactivation defective E2F-1 mutants [E2F-l(1-374) and E2F-1(1-284)] induced apoptosis at least as efficiently as wild-type E2F-1, although the non-DNA-binding E2F-1 (E132) mutant failed to induce apoptosis (Fig. 3A). Comparable levels of expression of each E2F-1 protein was seen under conditions of serum starvation (Fig. 3B). In light of the unexpected apoptotic activity of the two transcriptionally inactive E2F-1 mutants, we repeated the analysis in cells maintained in the presence of serum, conditions that had markedly reduced the apoptotic potential of wild-type E2F-1 (Fig. 1C). The ability of E2F-1 to promote cell cycle progression was also evident in the presence of serum and as seen in serum-deprived cells, all three transcriptionally inactive mutants failed to accelerate cell cycle progression and induced some degree of cell cycle arrest in the presence of serum (Fig. 3C,D). Although addition of serum significantly reduced wild-type E2F-1 mediated apoptosis, serum had only a modest effect on the cell death induced by E2F-l(1-374) or E2F-l(1-284). Because the transactivation deficient mutants were slightly more stably expressed than the wild-type protein (Fig. 2C), we analyzed the effect of transfecting 5 gg of the wild-type E2F-1 expression plasmid into cells grown in the presence of serum. Under these conditions, wild-type E2F-1 expression was increased to levels equivalent to that seen with the two mutants, but apoptosis did not increase to match the levels seen with E2F-l(1-374) or E2F-1(1-284) (data not shown). This suggests that the differences in apoptotic potential of wild-type E2F-1 and the trans-activation mutants is not simply a reflection of differences in levels of protein expression, although the possibility remains that we are underestimating apoptosis induced by wild-type E2F-1 by including cells dying from G2 within the S-phase population. This is less likely to occur in cells expressing the mutants because they are predominantly in G1. The DNA-binding defective E2F-1 (E132) mutant showed no apoptotic activity in any assay. The consistent failure of this mutant to induce apoptosis in either the presence or absence of serum indicated that the apoptotic function of the other mutants is also not simply a reflection of the titration of an E2F-l-binding protein such as DP1 or cyclin A, as a result of overexpression. Essentially identical data were obtained in H1299 cells (data not shown).

Ceil death induced by E2F-1 is apoptosis
In view of the unexpected apoptotic function of the E2F mutants, we sought to confirm that the cell death seen was a result of apoptosis. A number of lines of evidence strongly indicate that this is the case. Coexpression of the apoptosis inhibitor Bcl-2 resulted in the inhibition of cell death induced by both wild-type E2F-1 and the E2F-1(1-374) mutant (Fig. 4A). Western blot analysis of these cells showed that the protection afforded by Bcl-2 expression was not the result of a reduction in E2F-1 protein expression in the cotransfected cells (Fig. 4B). Examination of transfected cells morphology by DAPI staining revealed nuclear blebbing and apoptotic bodies in a significant number of E2F-l(1-374) expressing cells in the presence of serum (data not shown). A TUNEL assay, which measures the presence of nicked DNA, an early event in apoptosis, showed that a significantly higher proportion of cells expressing E2F-1(1-374) were TUNEL positive, compared to either E2F-1 or E2F-I(E132) expressing cells (Fig. 4C). These results show that the cell death generated by ectopic expression of E2F-l(1-374) shows morphological, biochemical, and biological features of apoptosis.

Cyclin A binding is not necessary for p53-independent E2F-I-induced apoptosis.
Previous studies have shown that loss of negative regulation of E2F DNA-binding activity by cyclin A associated kinase results in the inability of cells to leave Sphase and eventual apoptosis (Krek et al. 1995). Because this apoptotic function depends on the ability of E2F-1 to drive DNA synthesis, it is dependent on the presence of the transcriptional activation domain and the transactivation-deficient E2F-1 mutants show loss of this activity. The E2F-1 apoptotic activity described here is clearly distinct from the previously described function, and we were interested to determine whether the interaction of cyclin A with E2F-1 could also modulate the present apoptotic function. Analysis of E2F-1 mutants deleted of the cyclin A-binding site (E2F-l&24) (Krek et al. 1995) ( Fig. 2A), which showed enhanced apoptotic activity at the end of S-phase, behaved indistinguishably from the wild-type protein in the present assay (Fig. 5A), indicating that loss of regulation of E2F-1 by cyclin A does not contribute to the apoptotic function being measured here. Conversely, loss of the cyclin A-binding domain did not significantly reduce the apoptotic activity of the transactivation domain deleted E2F-1 mutant [E2F-1A24(1-374)], showing that cyclin A binding is not necessary for this function. Protein analysis showed similar expression levels for all the E2F-1 proteins in the transfected cells (Fig. 5B), although slightly enhanced expression of the mutants lacking the carboxy-terminal destabilization domain was seen again.
The expression of several genes with roles in regulating entry and progress through DNA synthesis has been shown to be regulated by E2F, including B-myb (Lam and Waston 1993), cyclin E (Ohtani et al. 1995), and cyclin A (Schulze et al. 1995). The apoptotic activities of the E2F-1 mutants indicated that transcriptional activation is not necessary to induce cell death, but the loss of function displayed by the DNA-binding mutant sug- gested that relief of transcriptional repression through E2F-binding sites may play a role. Transactivation domain deletion mutants of E2F-1 have been shown to retain the ability to block E2F-l-mediated transcriptional repression (Qin et al. 1995). Transcriptional repression through E2F-binding sites has been described for several cell promoters, including the B-myb promoter (Lam and Waston 1993) and the cyclin A promoter, which is repressed during the early stages of the cell cycle through a variant E2F-binding site (Schulze et al. 1995). Although any of these genes may be downstream mediators of the E2F-1 apoptotic response seen here, preliminary analysis of the consequence of ectopic cyclin A expression in these cells did not reveal any apoptotic activity, although accelerated cell cycle progression could be detected (data not shown).

D i s c u s s i o n
Our analysis of E2F-1 apoptotic function has shown that p53 is not essential for an E2F-1-induced apoptotic signal, and that the apoptotic activity of E2F-1 can be dissociated from the ability to accelerate cell cycle progression. The contribution of p53 to E2F-1-mediated apoptosis has been well established and we have also observed elevated apoptosis in cells transfected with p53 and E2F-1. The evidence is compelling that in some cell types in vivo, p53 plays an important role mediating this function of E2F-1. Nevertheless, the p53-independent apoptotic activity of E2F-1 described here is consistent with several previous studies that have documented a growth inhibitory effect of E2F-1 in similar p53 negative cells (Melillo et al. 1994;Qin et al. 1994;Berry et al. 1996). Furthermore, recent analyses of Rb-/-mice in p53-/-or +/+ backgrounds have clearly demonstrated p53 independent apoptosis mediated by loss of pRB in some tissues (Macleod et al. 1996). Our experiments suggest that the p53-independent apoptosis seen in this mouse model is mediated by E2F-1, and that this apoptotic activity of E2F-1 is physiologically important. The elimination of p53 from our experiments has also allowed a simpler dissection of the mechanisms by which E2F-1 activates cell death in the absence of a p53-mediated cell cycle arrest.
Although conflicting signals for cell cycle arrest and cell cycle progression may activate an apoptotic response, we show here that E2F-1 can exhibit separable cell cycle progression and apoptotic activities. Cell cycle progression and apoptotic functions have also been dissociated for the transcription factor c-Myc, where c-Myc induced apoptosis can be separated from c-Myc induced proliferation (Harrington et al. 1994;Packham et al. 1996;Rudolph et al. 1996). Similarly, p53 induced cell cycle arrest is separable from p53 apoptotic activity (Rowan et al. 1996), and although p53 can cooperate with E2F-1 in the induction of apoptosis, we have found that activation of a cell cycle arrest by direct expression of the p53 target p21WAF1/CIP1 does not enhance E2F-1-induced apoptosis (A.C. Phillips, unpubl.). Although the apoptotic function of E2F-1 does not appear to depend on E2F-l-dependent transcriptional activation, the inactivity of the E2F-l(E132) mutant indicates the importance of DNA binding. The presence of E2F-binding sites within some promoters has been shown to be respon- sible for repression of transcription at certain stages of the cell cycle, mediated by binding of E2F/pocket protein complexes (Lam et al. 1994;Sellers et al. 1995;Weintraub et al. 1992Weintraub et al. , 1995. The importance of DNAbinding activity to the E2F-1 apoptotic function described in this study suggests that this apoptotic activity may reflect the alleviation of E2F-mediated transcriptional repression, rather than the activation of E2F responsive genes. In support of this suggestion, the accompanying paper by Hsieh et al. shows that pRB can inhibit the apoptotic function of wild-type E2F-1, but not the E2F-l(1-374) mutant that fails to bind pRB. It should be pointed out, however, that most of our studies were carried out in Saos-2 cells, which fail to express wild-type pRB. Although this tumor cell line may have acquired additional alterations that protect it from the apoptotic activity of the endogenous E2F-1, the observation that enhanced expression of E2F-1 can induce apoptosis in these cells suggests that, under some circumstances, repression of the apoptotic target genes may also be carried out by E2F complexes containing other pocket proteins such as p107 and p130.
The expression of several cellular genes has been shown to be repressed through E2F-binding sites and any of these may be apoptotic targets for E2F-1 transcriptional activation. We have noted that expression of cyclin A can show an ability to promote cell cycle progression similar to that seen for E2F-1 without inducing apoptosis, further supporting the model in which the apoptotic function of E2F-1 is not simply a consequence of activation of cell cycle progression. Nevertheless, previous studies with p53 have shown that protein levels can be of critical importance in determining whether a cell undergoes cell cycle perturbations or apoptosis (Chen et al. 1996) and we cannot rule out an apoptotic activity of cyclin A. It is possible that several E2F-1regulated genes contribute to the apoptotic response, and we are generating cell lines with inducible E2F-1 in which to identify the critical target genes. It is of interest, however, that activation of cyclin-dependent kinases and induction of cyclin A by c-Myc has been shown to be independent of the Myc-induced apoptotic signal (Rudolph et al. 1996).
Failure to exit S phase, through phosphorylation and inactivation of E2F activity by cyclin A dependent kinase, has been shown to lead to apoptosis in rodent fibroblasts, and this activity requires the transactivation domain in E2F-1 (Krek et al. 1995). In contrast, the apoptotic activity of E2F-1 described in this study does not appear to depend on the ability of E2F-1 to induce Sphase progression and we would therefore like to propose that E2F-1 can generate an apoptotic signal at more than one stage of the cell cycle. The apoptotic function described here appears to be suppressed under conditions of normal cell growth, only becoming evident following a perturbation of normal cell cycle progression. In our model (Fig. 6), release of free E2F-1 from pocket protein interactions (either by ectopic overexpression or normal cell cycle progression) simultaneously activates expression of genes necessary for DNA synthesis and dere- presses expression of the apoptotic genes. Under conditions that allow progress into S phase, the apoptotic signal is suppressed. However, the apoptotic activity is revealed in the absence of normal cell cycle progression, either following serum starvation or expression of transactivation domain E2F-1 mutants, which prevent activation of the appropriate S-phase-specific genes while retaining the ability to derepress expression of the apoptotic targets (Qin et al. 1995). The DNA-binding mutant fails to induce apoptosis because it is defective for both activation of S-phase genes and derepression of apoptotic genes. According to this model, ectopic expression of pRB would result in maintenance of transcriptional repression of the apoptotic targets, although the apoptotic activity of E2F-1 in pRB-deficient Saos-2 cells clearly complicates the interpretation of the results. Nevertheless, our model would predict a function for E2F-1 in monitoring conditions of cell cycle progression, and activation of an apoptotic function in response to perturbations of normal cell proliferation is consistent with the tumor suppressor activity of E2F-1.
p53 expression was driven from the MoLTR in the construct pJ4f~p53 (Crook et al. 1994b), human Bcl-2 cDNA was cloned into pcDNA3 (Invitrogen) for expression from the CMV promoter. Where appropriate, transfection efficiency was monitored by use of pJ4f~gal, a plasmid in which expression of the ~-galactosidase gene is under the control of the MoLTR (Crook et al. 1994a). For detection of E2F-1, the monoclonal antibody KH20 was used . CD20 expression was detected by use of FITC conjugated anti-CD20 monoclonal antibody (Becton Dickinson), p53 expression was detected by use of the monoclonal antibody DO-1 (Vojtesek et al. 1992) and Bcl-2 expression was detected with a polyclonal rabbit antiserum E2F-1 ~ --~ II= CAPOPTOSIS~ T T E2.-1 (1~,,i ~ (~CELL CYCLE'~ I r ~ROGR~SSIONJ Growth no s~um signals Figure 6. Model of E2F-1 function. E2F-1 activates transcription of genes necessary for cell-cycle progression and de-represses expression of apoptotic genes. In the presence of growth signals (serum), normal cell-cycle progression suppresses the apoptotic signal. Failure of normal cell-cycle progression, either through loss of growth signals or inability to activate E2F-1 responsive cell-cycle progression genes, results in the manifestation of the apoptotic response.
Cold Spring Harbor Laboratory Press on July 22, 2018 -Published by genesdev.cshlp.org Downloaded from raised against a Bcl-2 peptide corresponding to amino acids 41-54.

Cell culture and transfection
Saos-2 and H1299 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. For transient transfection, 4 x 105 (for reporter gene assays) or 8 x l0 s [for fluorescence-activated cell sorting (FACS)analysis] were seeded onto 10 cm dishes and transfected the following day, by use of calcium phosphate coprecipitation. Unless otherwise stated, all transfections were cotransfections of 1 ~g test plasmid with 0.5 ~g pCMVCD20, and the transient transfection efficiencies were within the range of 5-10%. Cells were harvested after 24-72 hr and prepared for flow cytometry or luciferase and ~-galactosidase assays. In each case, one fifth of the sample was retained for protein analysis.

Transcriptional activity and protein analysis
Cell lysates were prepared 24 hr after transfection and analyzed for luciferase and ~-galactosidase activity as described previously (Lain and Waston 1993). For Western blotting, proteins in equal quantities of lysates were separated by 12.5% polyacrylamide gel electrophoresis and transferred overnight onto nitrocellulose filters.

Flow cytometric analysis
For flow cytometry, total populations of floating and adherent cells were harvested 24-72 hr post-transfection, stained for CD20 expression, fixed in ethanol and stained for DNA content as described previously (Zhu et al. 1993). Cells stained directly for E2F-1 expression were fixed first and subsequently stained as described previously (Rowan et al. 1996). Flow cytometric analysis was carried out (FACScalibur, Becton Dickinson) and total cell populations were gated and analyzed as described previously (Rowan et al. 1996). Briefly, cells were measured for their FITC intensity (green channel) and propridium iodide fluorescence (red channel). Total populations were gated to remove doublets and very small debris. Background levels of FITC fluorescence were established by use of vector only transfected cells and CD20 or E2F-1 expressing cells were gated and analyzed for cell cycle distribution. TUNEL staining was carried out with the ApopTag Direct kit (Oncor) according to the manufacturer's instructions.