Studies employing Saccharomyces cerevisiae cpt1 and ept1 null mutants implicate the CPT1 gene in coordinate regulation of phospholipid biosynthesis.

The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding sn-1,2-diacylglycerol choline phosphotransferase and sn-1,2-diacylglycerol choline/ethanolamine phosphotransferase, respectively. Incorporation of 32Pi into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in wild type and ept1 strains was decreased in the presence of exogenous inositol. In contrast, inositol did not affect 32Pi incorporation into phospholipid in cpt1 or cpt1ept1 strains. In membranes isolated from wild type and ept1 strains grown in the presence of inositol or inositol/choline, the CPT1-derived cholinephosphotransferase activities were reduced 40-50 and 65%, respectively. Inositol-dependent reductions in CPT1 derived choline-phosphotransferase activity correlated with transcript levels in both wild type and ept- backgrounds. The ethanolaminephosphotransferase activity of the EPT1 gene product in wild type cells was reduced 40% by exogenous inositol alone and 50% by inositol/choline. In the cpt1 strain, however, the ethanolaminephosphotransferase activity was unaffected by exogenous inositol or inositol/choline. The inositol-dependent reduction of ethanolaminephosphotransferase activity observed in wild type cells correlated with reduced levels of EPT1 transcripts; in the cpt1 strain, EPT1 transcript levels were not affected by inositol. These results indicate that 1) a functional CPT1 gene or gene product is required for inositol-dependent regulation of phospholipid synthesis; 2) the enzyme activities of both the CPT1 and EPT1 gene products are repressed by inositol and inositol/choline, and require an intact CPT1 gene; 3) inositol mediates its regulatory effects on phospholipid synthesis via a transcriptional mechanism.

In the yeast Saccharomyces cereuisiae, the predominant route for PC and PE biosynthesis is thought to be from phosphatidylserine (PS) (2). In the PS-dependent pathway, PS, synthesized from CDP-diacylglycerol and serine by PS synthase, is decarboxylated to PE which, in turn, is methylated to PC (Fig.  1). However, the contributions of the Kennedy pathway to overall synthesis of PC and PE in yeast is not well understood. Recently, it has been shown that mutations in the Kennedy pathway of PC synthesis are able to bypass mutations in SEC14 (4), a gene encoding a Golgi-associated yeast PCP1 transfer protein (5). This observation, suggests the possibility that the Kennedy pathway of PC synthesis may play an important role in the coupling of phospholipid synthesis and the protein secretory pathway.
Although little is known on the coordinate regulation of phospholipid synthesis in mammalian cells, considerable understanding of the regulation of phospholipid biosynthesis in yeast has been achieved (reviewed in Ref. 6). This regulation is mediated by the availability of the water-soluble phospholipid precursors choline and inositol. A number of yeast phospholipid biosynthetic enzymes, including CDP-diacylglycerol synthase (71, PS synthase (81, PS decarboxylase (9, lo), the PE N-methyltransferases (11,121, and choline kinase (13) are repressed in cells grown in the presence of inositol. Inositol-l-phosphate synthase, which catalyzes the first step in PI biosynthesis, is also repressed by inositol (14). Addition of choline to medium containing inositol results in a further repression of these activities. The structural genes for PS synthase (CHOl), choline kinase ( C H I ), inositol-l-phosphate synthase (IN01 ), and the PE N-methyltransferases (CHO2IPEMl) and OPZ3 IPEM.2) have been cloned, and the observed repression of these enzymes by inositol and inositolkholine was shown t o occur at the level of mRNA abundance (13,15,16).
Two classes of regulatory mutants have also been identified. The in02 and in04 mutants (inositol auxotrophs) show constitutive repression of the activities that are regulated in response to inositol and choline in wild type cells (15)(16)(17). In contrast, opil mutants (7, 8, 15,16) constitutively express these same activities and display an inositol excretion phenotype. It has been observed that cells which are decreased in the synthesis of PC are not regulated by inositol and exhibit an opil phenotype (6,181, suggesting that ongoing PC synthesis is required in the coordinate regulation of phospholipid metabolism mediated by inositol. Our laboratory has cloned two yeast structural genes for sn-1,2-diacylglycerolaminoalcoholphosphotransferases: the CPTl gene which encodes a cholinephosphotransferase (19) and the EPTl gene whose product possesses both choline-and ethanolaminephosphotransferase activities (20). Strains bearing cptl and eptl null mutations as well as a cptl eptl double null mutant (21) were constructed by chromosomal disruption. In the present work, these strains were employed to investigate the roles of the CPTl and EPTl genes in yeast phospholipid synthesis, to evaluate the effect of exogenous inositol and choline on the expression of their gene products, and to explore the  (19). Safety-Solv was from Research Products International. Amino acids, myo-inositol, phenol, salmon sperm DNA, phospholipase C (Bacillus cereus), bovine serum albumin, choline chloride and MOPS were purchased from Sigma. Dioleoylphosphatidylcholine and dipalmiteoylphosphatidylcholine were obtained from Avanti Polar Lipids, Inc.; the corresponding diacylglycerols were prepared from them by phospholipase C digestion (22). Triton X-100 was obtained from Pierce. Oligo(dT)-cellulose was purchased from Collaborative Research, Inc. Zeta-probe cationic membranes, SDS, and agarose were from Bio-Rad. Restriction enzymes, RNA ladders, formamide, and DNA polymerase I (Klenow fragment) were obtained from Life Technologies, Inc. Materials for growth media were purchased from Difco.
Equilibrium-labeling Studies-Yeast cultures (10 ml) were grown overnight (minimum of five doublings) on minimal media at 30 "C with ',Pi (0.4 mCi/mmol) in the absence or presence of 1 m M choline, 50 1.1~ inositol, or both choline and inositol. Cells were harvested at midlogarithmic growth phase (OD,, of 0.5-0.8) by centrifugation at 2500 x g for 5 min, washed with 5 ml of ice-cold water, resuspended in 1 ml of methanol, 1 M NaCl (5/4), and transferred to a 1.5-ml beadbeater vial containing 0.75 ml of 0.5-mm glass beads. Cells were broken by three 1-min bursts on a BioSpec Products mini-beadbeater with cooling on ice between bursts. The homogenate was removed to a 16 x 125-mm glass tube containing 4 ml of chloroform and the beads rinsed three times with 1 ml of methanol, 1 M NaCl. The final ratio of chloroform/ methanoY1 M NaCl was 2:l:O.g (v/v). The lower organic phase was washed twice with ideal upper phase (methanoY1 M NaCVchloroform; 1:1:0.06 (v/v)), dried under nitrogen, and resuspended in 1 ml of chloroform. Aliquots were removed for liquid scintillation counting, thin layer chromatography (TLC), and quantitation of phospholipid phosphorus by the method of Ames and Dubin (23). Phospholipids were separated by two-dimensional TLC on Silica Gel G-60 plates (Merck). Separation in the first dimension was in chloroform/methanol/30% NH,OWwater (66:27:3:0.8, v/v) and in the second dimension with chlorofodmethanoYacetic acid/water (32:4:5:1, v/v) (24). Radioactive spots were located by autoradiography using Kodak X-Omat AR film, scraped into scintillation vials, and counted in a LKB 1217 Rackbeta liquid scintillation counter. Lipids were identified by comparison to authentic standards.
Pulse-labeling Studies-Cells were cultured and extracted as described above with the exception that 32Pi (5.4 mCi/mmol) was added to growth media 15, 30,45 and 60 min prior to harvesting. Phospholipids were separated by one-dimensional TLC in chlorofodmethanoYacetic acid/water (60:50:1:4, v/v) or chloroform/methanol/ammonium hydroxide/water (70:30:4:2, v/v), visualized by autoradiography or iodine staining, and either scraped into scintillation vials for counting or analyzed using a Bioscan System 200 imaging scanner.
Isolation of Membranes-Yeast cultures (500 ml) were grown in minimal media in the absence or presence of 1 m~ choline or 50 ph! inositol or both to mid-logarithmic phase and harvested as described previously (19). Following glass bead disruption, the homogenate was removed, beads were rinsed three times with 0.5 ml of GME (20% glycerol, 50 m M MOPSNaOH, pH 7.5, 1 m~ EDTA) and the homogenate and washes centrifuged 10 min at 16,000 x g. The resulting supernatant was centrifuged at 100,000 x g for 15 min in a Beckman TL-100 ultracentrifuge. The membrane pellet was suspended in 0.5 ml of GME with the aid of a glass-Teflon homogenizer and aliquots were stored at -70 "C. Membrane protein was measured by the method of Peterson (25) using bovine serum albumin as standard.
Mixed Micellar Assay-The choline-and ethanolaminephosphotransferase activities of isolated yeast membranes were determined by incorporation of label from [32P]CDP-choline or [32PlCDP-ethanolamine, into PC and PE, respectively, using a Triton X-100-mixed micellar assay as described previously (21). The assay contained in a total volume of 0.2 ml: 50 m M MOPSNaOH, pH 7.5, 20 m~ MgCl,, 0.45% Triton X-100 (6.5 mM), 10 mol % dioleoyl-PC, 10-20 pg of membrane protein, and either 5 mol % dioleoylglycerol and 100 1.  Isolation and Northern Analysis of RNA-Yeast cultures (500 ml) were grown to mid-logarithmic phase as described above. Total cellular RNA was isolated by glass bead disruption followed by hot phenol extraction as described by McAlister and Finkelstein (26). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography according to the manufacturer's directions. RNA was quantitated by measuring absorbances at 260 nm, using a n extinction coefficient of 40 pg/ml (27), and stored in water at -70 "C.
DNA probes were radiolabeled to high specific activity (10' counts/ midpg) with [32P]dCTP by the oligolabeling method (28,291 and used at 2-5 x lo6 countdmidml of hybridization solution. Prehybridization and hybridization of radiolabeled probes were performed at 42 "C in 50% formamide, 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 x Denhardt's (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% Ficoll), 0.5% SDS, and 100 pg/ml of salmon sperm DNA (27). Following hybridization, membranes were washed for 15 min each with 2 x SSC, 0.1% SDS and 0.5 x SSC, 0.1% SDS at room temperature and with 0.1 x SSC, 0.1% SDS at 65 "C. Washed membranes were exposed to Kodak X-Omat AR film a t -70 "C with a n intensifying screen. Membranes were stripped prior to rehybridization by heating at 95 "C in an excess of 0.1 x SSC, 0.5% SDS for 20 min. RNA was quantitated using a LKB Ultrascan XL laser densitometer. Only measurements which were within the linear limits of the film and densitometer were used.

RESULTS AND DISCUSSION
Equilibrium-labeling Studies-To investigate the roles of the CPTl and EPTl gene products in phospholipid metabolism, the growth characteristics and phospholipid composition of mutant strains bearing null mutations in the CPTl and EPTl loci were compared to the parental wild type strain. The response of cptl and eptl mutants to the presence of choline (1 mM) and inositol (50 p) in the growth medium was also assessed. The growth characteristics of all strains were similar (doubling time of 3 h) and were unaffected by addition of choline or inositol.
Phospholipid compositions were determined by labeling to constant specific activity with 32Pi (0.4 mCi/mmol) for a minimum of five generations. Under these conditions, the specific activity of [32P]phospholipid was constant at 868 2 30 counts1 midnmol phospholipid phosphate for all strains regardless of media additions. These results indicate mutations in cptl and eptl have no effect on overall cell growth or amount of total phospholipid.
To assess the effect of cptl and eptl mutations on phospholipid class distribution, phospholipids were separated by twodimensional thin layer chromatography and the percentage of label in each phospholipid determined (Table I). No major differences in phospholipid compositions were observed between wild type and mutant strains, indicating any perturbations in phospholipid synthesis resulting from mutations in the CPTl and EPTl loci are largely compensated in the steady-state. However, some significant, albeit small changes, in phospholipid composition were observed between strains (Table I).
PC levels were higher in the eptl mutant compared to other strains but were similar to wild-type in the presence of inositol. This increase was not observed in the double null mutant in which the synthesis of PC was also compromised, suggesting higher levels of PC in the eptl strain may result from an increased availability for the CPTl gene product of the common substrate, sn-l,2-diacylglycerol. Consistent with this observation is the recent findings of this laboratory that have determined that the CPTl gene product contributes to 95% of PC synthesized via the CDP-choline pathway, while the EPTl gene product constitutes the remaining 5% (30). Inositol supplementation resulted in increased levels of PI and decreased PC in all strains. PS levels were appreciably reduced in the cptl strain in the presence of inositol and to a lesser extent in the cptl eptl strain, suggesting alterations in lipid metabolism induced by mutations in cptl are alleviated by simultaneous mutation in eptl.
Pulse-labeling Studies-While the effect of cptl and eptl mutations on phospholipid distribution in the steady-state were small, differences observed in the presence of exogenous inositol suggested some alterations in phospholipid regulation in these strains. Pulse-labeling studies with 32Pi were undertaken to determine the rate of synthesis of phospholipids in each strain in the absence and presence of the water-soluble precursors, choline and inositol (Fig. 2). In the absence of exogenous precursors or in the presence of choline alone, no differences were observed in the incorporation of 32Pi in wild type and eptl strains during a 1-h labeling period. Under the same conditions, incorporation of label into phospholipids in the cptl and cptl eptl mutants were increased slightly over wild type. In the  presence of exogenous inositol, the total incorporation of label into phospholipids was reduced 2.5-fold in wild type and eptl strains. In contrast, label incorporation was not reduced by inositol in either strain bearing a cptl null mutation, suggesting that a functional CPTl gene or gene product is essential to observe this effect of inositol on phospholipid metabolism. Analysis of individual phospholipids revealed that reduced incorporation of radiolabeled phosphorus in wild type and eptl strains in the presence of inositol was primarily attributable to decreased synthesis of PC (Fig. 3). PC synthesis was decreased 65-75% in wild type and eptl strains in the presence of inositol. The synthesis of PS and PE were also reduced in these strains in the presence of inositol while the synthesis of PI was relatively unchanged. Exogenous choline stimulated the synthesis of PC in wild type and eptl mutants, most likely due t o increased flux through the Kennedy pathway of synthesis.
In both strains bearing a cptl null mutation, PC synthesis was lower than wild type or eptl strains in the absence of exogenous precursors. If PC is synthesized solely by the PSdependent pathway in the absence of exogenous choline, then no difference between wild type and cptl strains would be expected. The observed differences suggest that the Kennedy pathway may contribute significantly to PC synthesis in the absence of exogenous precursors, presumably by utilization of choline derived from turnover of PC synthesized by the PSdependent pathway. Alternatively, mutations in the CPTl locus may have pleiotropic effects on the enzymes of the PS-dependent pathway of PC synthesis.
In the cptl and cptl eptl strains, neither PE nor PS synthesis was reduced by growth in inositol as was observed for strains possessing an intact CPTl locus. Inositol supplementation increased PI synthesis 3-fold in the cptl strain and 1.7-fold in cptl eptl strain which exhibited higher levels of synthesis in the absence of exogenous inositol. These findings were in contrast to strains lacking a cptl mutation where PI synthesis remained constant under the growth conditions examined. The cptl eptl double null strain exhibited higher levels of PS and PI synthesis compared to the other strains under all growth conditions. This increase may reflect a shift in phosphatidic acid metabolism from diacylglycerol synthesis in the absence of functional aminoalcoholphosphotransferases and toward CDP-diacylglycerol synthesis, the common precursor of both PS and PI (Fig. 1).
Results of the pulse-labeling studies indicated dramatic effects of mutations in the CPTl locus on both the class distribution of phospholipid synthesis and changes in the pattern of synthesis induced by inositol. Taken together, these observations suggest the participation of the CPTl gene product in PC synthesis independent of the availability of exogenous choline and that a functional CPTl gene or gene product is required for inositol dependent regulation of phospholipid synthesis.
Regulation ofActivities of the CPTl and EPTl Gene Products by Choline and Inositol-Inositol pleiotropically represses the expression of several enzymes of phospholipid biosynthesis at the level of transcription (13,15,16). To extend these observations to encompass the Kennedy pathway enzymes, the activities of the CPTl and EPTl gene products were compared in membranes isolated from wild type, cptl, and eptl strains grown in the absence or presence of choline and inositol (Table  11). Activities were measured in a mixed-micelle assay (21) containing Triton X-100 (0.45%), 10 mol % dioleoyl-PC as phospholipid activator, and either 5 mol % dioleoylglycerol for the EPTl gene product or 5 mol % dipalmiteoylglycerol for the CPTl gene product. Previous results from our laboratory have shown that the CPTl gene product is primarily a cholinephosphotransferase, while the EPTl gene product catalyzes cholineand ethanolaminephosphotransferase reactions (19,20). In order to independently assess the CPTl-dependent cholinephosphotransferase activity in wild type strains containing a functional EPTl gene product, the preference of the CPTl gene product for dic,,,, over dic,,,, was exploited.* As shown in Table 11, the presence of exogenous choline had little effect on either choline-or ethanolaminephosphotransferase activities. In wild type cells grown in the presence of While the EPTI gene product activities exhibited relative specificity for diC18:, compared to diCIG:l, the CPTl gene product dependent CPTl activity displayed a preference for diC,,:, (S. C . Morash, R. H. Hjelmstad, C. R. McMaster, and R. M. Bell, unpublished observations). The EPTl gene product-dependent cholinephosphotransferase activity represents at most 15% of the total CPTI activity in wild type membranes; therefore, the diC,,,,-dependent cholinephosphotransferase activity is essentially a measure of the CPTl gene product activity. inositol, the CPTl-dependent cholinephosphotransferase activity was reduced approximately 50%, and the EPTl-dependent ethanolaminephosphotransferase activity was reduced 40%. When both choline and inositol were present in the growth medium, CPTl-dependent cholinephosphotransferase activity and EPT1-dependent ethanolaminephosphotransferase activity were reduced 65 and 53%, respectively. These reductions correlated with the lower synthetic rates of PC and PE observed in the presence of inositol in the labeling studies (Fig. 3).
In the eptl strain, cholinephosphotransferase activity is due solely to the CPTl gene product. This activity was slightly higher compared to wild type and exhibited comparable levels of reduction by inositol and inositollcholine. In contrast, neither the choline-nor ethanolaminephosphotransferase activities of the EPTl gene product were affected by exogenous inositol or inositollcholine in the cptl strain.
Thus both the CPTl and the EPTl gene product activities were repressed in the presence of inositol and inositoVcholine; the observed degrees of repression were quantitatively comparable to those previously reported for other enzymes of phospholipid synthesis (7)(8)(9)(10)(11)(12)(13)(14). Changes in EPTl gene product activity in response to inositol and inositollcholine were dependent on the presence of an intact CPTl locus. Constitutive expression of the EPT1-dependent activity in the cptl strain correlated well to the higher rates of PE synthesis observed in the cptl strain in the presence of inositol (Fig. 3).
7kanscriptional Regulation of CPTl and EPTl mRNA Levels-Northern analysis was undertaken to determine whether the observed repression of the CPTl and EPTl gene product activities by inositol and inositolkholine in wild type and eptl strains occurred at the level of transcription. Levels of EPTl-and CPTl-hybridizing RNA (Figs. 4 and 5, respectively) were assessed in wild type, cptl, and eptl strains grown in the absence or presence of choline and/or inositol. In wild type cells, the 1.4-kb EPTl-derived transcript (Fig. 4) was reduced 70% in the presence of inositol and 80% in inositol plus choline compared to the no addition media. Significantly, this reduction in EPTl transcript was not observed in the cptl strain. These results strongly suggest that the reduction of ethanolaminephosphotransferase activity in wild type cells grown in the presence of inositol is a consequence of transcriptional regulation of the EPTl gene and that this regulation requires an intact CPTl gene.
In wild type and eptl strains, a 1.4-kb CPT1-derived transcript was expressed regardless of growth supplementation (Fig. 5A). The levels of this transcript were unaffected by the addition of choline but were decreased in the presence of inositol or inositoYcholine (Fig. 5 B ) (30). The changes in transcript levels in response to choline and inositol supplementation were consistent with the changes in CPT1-derived cholinephosphotransferase enzyme activities (Table 11). A second 1.7-kb CPTlderived transcript was induced in the presence of inositol and inositolkholine (Fig. 5A). The nature of this induced transcript or its role in phospholipid metabolism is unknown. However, since the CPT1-dependent cholinephosphotransferase activity is reduced in wild type and eptl strains grown in the presence

I1
Choline-and ethanolaminephosphotransferase activities of membranes isolated from wild type, cptl, and eptl strains dures." Cholinephosphotransferase activity was measured using 250 VM t32PlCDP-choline and either dipalmiteoylglycerol (diC16:l) or dioleoylglyc-Membranes from each strain grown under the four indicated conditions were prepared and assayed as described under "Experimental Proceerol (diC18:l). The ethanolaminephosphotransferase activity of the EPTl gene product was assayed with 100 p~ t32PlCDP-ethanolamine and diCIBl. In the wild type strain which contains both CPTl and EPTl gene product activities, the diC,,,,-dependent choline phosphotransferase activity is essentially an independent measure of CPTZ-dependent activity (see Footnote 2). Results are the means t S.D. from three separate membrane inositol were added to the growth media at 1 m M and 50 PM, respectively.
preparations assayed twice (n = 6). The activities are also expressed as a percentage relative to the no addition medium (parenthesis  * Not detected. of inositol (Table 111, the induced 1.7-kb transcript is unlikely to support the translation of an enzymatically functional CPTl gene product. Detailed analysis of the precise origin of the induced transcript and its putative role in the regulation of phospholipid metabolism will be required to definitively address this issue. Concluding Discussion-The PS-dependent and Kennedy pathways in yeast have traditionally been regarded as alternative routes for synthesis of PC and PE and have been referred to as the primary and auxiliary pathways, respectively (6, 18). The present studies provide new information which requires reconsideration of existing conceptions regarding the relationship between these two pathways. Reduced synthesis of PC and PE in the cptl and eptl strains compared to wild type (Fig. 3) suggests significant synthesis of these phospholipids via the Kennedy pathway in the absence of exogenous choline or ethanolamine. While endogenous ethanolamine is known to derive from sphingolipid metabolism (311, there are no known endogenous sources of de novo choline synthesis in yeast. Thus, it is possible that synthesis of PC via the Kennedy pathway utilizes choline derived from PC turnover in the absence of exogenous choline (32). The role of phospholipid turnover in generating substrate pools for phospholipid resynthesis and the regulation of this process has not been studied. It seems likely that phospholipid turnover may also be an important factor in the effect of inositol on patterns of phospholipid synt h e~i s .~ The central role of inositol in the complex regulation of yeast dium did not effect growth rate or total phospholipid content in any Since the addition of inositol or inositoVcholine to the growth mestrain, the data in Fig. 2 demonstrates that inositol decreased both absolute rates of incorporation of 32Pi into phospholipid and the rate of approach to steady-state labeling. These results cannot be explained without inferring a simultaneous decrease in phospholipid degradation in inositol-grown cells. Preliminary equilibrium-chase labeling experiments have indicated such an effect of inositol on phospholipid degra- phospholipid synthesis is well documented and suggests that PC and inositol metabolism are coordinated (6, 18). The synergistic effect of choline on the repression of enzyme activities by inositol in the PS-dependent pathway of PC synthesis has been interpreted to represent a response to utilization of these precursors in the Kennedy pathway (6, 18). The finding that activities of the CPTl and EPTl gene products were regulated by exogenous inositol and inositolkholine and that the direction and magnitude of this regulation is similar t o that observed for enzymes of PS-dependent PC and PE synthesis does not support a model of alternate pathways. Thus, the PS-dependent and Kennedy pathways of PC and PE synthesis appear to represent complementary routes of synthesis. The overall regulatory effect of inositol and inositolkholine is to coordinately repress the biosynthetic enzymes for PS, PE, and PC. PI synthase (8) is unaffected by exogenous inositol, however, its substrates CDP-diacylglycerol (7) and inositol (14) are regulated by inositol. The only enzyme known to show increased activity in the presence of inositol is phosphatidic acid phosphatase which dephosphorylates phosphatidic acid to produce diacylglycerol (33). Taken together, these observations suggest that the regulatory effect of inositol is to coordinately decrease phospholipid synthesis and increase diacylglycerol synthesis. The increased diacylglcerol may be used for the synthesis of triacylglcerol as occurs when cells enter the stationary phase of cell growth (34).
The EPTl and CPTl genes are clearly shown in these studies to constitute a new addition to the group of genes subject to inositol repression. The regulatory effects of inositol are mediated through a 9-base pair repeated element (consensus 5'-ATGTGAAAT-3') that is present in the promotor regions of many phospholipid biosynthetic genes (including CPTl and EPTl ) and requires the activities encoded by the ZN02,ZN04, and OPZl regulatory genes (6, 18, 35, 36). It has been hypothesized that the regulatory system requires ongoing PC synthesis on the basis of misregulation of inositol-1-phosphate SFthase expression by inositol in mutants in the PS-dependent pathway. This misregulation is alleviated by the provision of precursors capable of being assimilated via the Kennedy pathway. In this model, a metabolic signal derived from the endproduct PC is envisioned (6, 18).
In the present work, two independent lines of evidence implicate the CPTl gene andlor gene product in mediating, at least in part, the regulation of phospholipid synthesis effected by inositol: 1) inositol-dependent alterations in patterns of phospholipid synthesis were not observed in strains bearing a cptl null mutation; and 2 ) regulation of EPTl transcription by inositol was absent in the cptl null mutant. The misregulation of EPTl expression in the cptl mutant suggests that other structural genes known to be subject to inositol-dependent repression may be misregulated in a cptl background.