CDP-choline and PtdEtn methylation pathways for PtdCho biosynthesis. Cho, choline; Cho-P, choline phosphate; CDP-Cho, CDP-choline; PtdMME, phosphatidylmonomethylethanolamine; PtdDME, phosphatidyldimethylethanolamine; SAM, S-adenosylmethionine; SAHC, S-adenosylhomocysteine.

Phospholipid profiles of wild-type and sac1 mutant yeast strains. (A) Yeast strains were pulse radiolabeled for 20 min with [³²P]orthophosphate at 26°C in inositol- and choline-supplemented medium. Radiolabeled phospholipids were extracted using an acidic extraction solvent suitable for recovery of phosphoinositides (see MATERIALS AND METHODS) and resolved by two-dimensional paper chromatography. Phospholipid species are numbered. Relevant phospholipids are identified as follows: 1, PtdIns-4-P; 4, PtdIns; 5, PtdSer; 6, PtdCho; 7, PtdEtn; 8, PtdOH. The spot 1 lipid also labels with [3H]inositol, and its assignment as PtdIns-4-P is justified below. The sac1-22 strain exhibited a dramatic elevation in both PtdIns-4-P and PtdCho and a significant reduction in PtdSer, relative to wild-type yeast. These aberrant levels of PtdCho, PtdIns-4-P, and PtdSer were typical of Δsac1 strains as well. Phospholipids extracted from the equivalent number of wild-type and sac1-22 cells were chromatographically resolved in this experiment. (B) Deacylation of [³H]inositol-labeled lipids from sac1 strains reveals a dramatic increase in a soluble species that coelutes with the glycerophosphoinositol 4-P standard (i.e. the deacylated product of PtdIns-4-P). Total cellular lipids prepared from equal cell equivalents of [³H]inositol-labeled wild-type (CTY182) or isogenic Δsac1 strain (CTY244) were deacylated, and resulting glycerophosphoinositols were analyzed by HPLC. The radioactive profile from the region corresponding to glycerophosphoinositol monophosphates is shown for CTY182 (dotted line, lower panel) and CTY244 (solid line, upper panel). The elution position of glycerophosphoinositol-4-P was identified by an internal ³²P standard included in HPLC analysis of deacylated fractions prepared from CTY244 (open circles, upper panel). In other experiments, radiolabeled glycerophosphoinositol, glycerophosphoinositol 4-P, and glycerophosphoinositol 4,5-P₂ were used as standards to further verify assignments. (C) Inositol polyphosphate 1-phosphatase digestion of soluble head groups derived by deglyceration of deacylated inositol lipids prepared from the Δsac1 strain CTY244. Reactants of control (upper panel) or inositol polyphosphate-1-phosphatase-treated (lower panel) deglycerated samples were separated by anion-exchange HPLC. Peaks corresponding to Ins-1-P, Ins-4-P, Ins-1,3-P₂, and Ins-1,4-P₂ eluted at 17, 18, 33, and 35 min, respectively.

Phosphatidylcholine synthesis in wild-type and sac1 mutant yeast strains. (A) Strains with the indicated genotypes (at bottom) were grown to midlogarithmic growth phase in medium containing inositol (0.1 mM) and choline (1 mM). Cell cultures were then pulse radiolabled with [³²P]orthophosphate (10 μCi/ml) for 30 min at 25°C. Phospholipids were extracted by the method of Atkinson (1984) and resolved by two-dimensional chromatography, and radiolabeled species were quantified by phosphorimaging (see MATERIALS AND METHODS). Biosynthetic rates were deduced from the relative proportion of label recovered in each individual phospholipid species (expressed as percentage of total phospholipid). Rates of total phospholipid synthesis in these strains were estimated by measuring incorporation of ³²P into chloroform-soluble counts (11,300–12,600 cpm/OD₆₀₀ for sac1 and sac1, Δspo14 mutants to 10,000–11,000 cpm/OD₆₀₀ for wild-type strains). Error bars are indicated. Phospholipid species are presented in the following order: PtdIns (black bars), PtdCho (cross-hatched bars), PtdSer (hatched bars), PtdEtn (stippled bars), and phosphatidic acid (open bars). Strains used were CTY1-1A (sec14-1^ts, SAC1, SPO14), CTY165 (sec14-1^ts, sac1-22, SPO14), and CTY1261 (sec14-1^ts, sac1-22, Δspo14). These data represent an average of three independent experiments, and all strains were analyzed in parallel for each trial. In a typical quantitation, PtdCho accounted for 5.0 × 10⁵ and 2.7 × 10⁵ phosphorimager units in sac1 and SAC1 strains, respectively, when 1 × 10⁶ phosphorimager units of total phospholipid were analyzed from each strain. (B) Activities of the CDP-choline pathway and PtdEtn methylation pathway were evaluated in vivo by pulse radiolabeling of SAC1 and sac1-22 strain pairs (CTY1-1A and CTY165, respectively). The indicated strains were grown to midlogarithmic growth phase in choline-free medium containing inositol. Cell cultures were split, and one aliquot was pulse radiolabeled with [¹⁴C]choline chloride (1 μCi/ml) or [¹⁴C]methyl-methionine (1 μCi/ml), respectively, for 20 min at 26°C with shaking. A parallel aliquot was labeled with [³²P]orthophosphate for 20 min at 26°C. Phospholipids from equal cell equivalents (determined by ³²P incorporation into phospholipid in the parallel cultures) were subsequently extracted, resolved, and quantified as described in MATERIALS AND METHODS. These data represent an average of three independent experiments. For quantitative comparison of a representative experiment, wild-type and sac1 strains incorporated 3.8 × 10⁴ and 1.3 × 10⁵ cpm [¹⁴C]choline chloride into PtdCho per OD₆₀₀ cells, respectively. (C) Strains with the indicatedgenotypes were grown to midlogarithmic growth phase in choline-free medium containing inositol. Cell cultures were split, and one aliquot was pulse radiolabeled with [¹⁴C]choline chloride (1 μCi/ml) or [³²P]orthophosphate (10 μCi/ml) for 20 min at 26°C. Phospholipids were extracted, resolved, and quantified (see MATERIALS AND METHODS). Again, samples were normalized for cell equivalents based on incorporation of [³²P]orthophosphate into the parallel cultures as described in B. These data represent an average of three independent experiments. Strains CTY182 (SAC1) and CTY244 (Δsac1) were transformed with the test YEp(DGK) plasmid and the vector-only control YEp(URA3) plasmid, respectively. The YEp(DGK) plasmid was represented by the pCTY85 construct that drives constitutive expression of DGK in yeast (Kearns et al., 1997).

Effects of PLD deficiency on PtdIns-4-P accumulation and bypass Sec14p. (A) Strains CTY1-1A (SAC1, SPO14), CTY165 (sac1-22, SPO14), and CTY (sac1-22, Δspo14) were grown in medium containing inositol (0.1 mM) and pulse radiolabeled with [³H]inositol (5 μCi/ml) for 1 h at 25°C and shaking. Radiolabeled inositol phospholipids were subsequently extracted and resolved by two-dimensional paper chromatography. Radiolabeled species were visualized by autoradiography. The positions of PtdIns and PtdIns-4-P are identified. These data are from a representative experiment. (B) The conditions of the experiment were exactly as in A with the exception that cells were radiolabeled with [³²P]orthophosphate (10 μCi/ml). Radiolabeled species were subsequently extracted under acidic conditions optimal for recovery of phosphoinositides, resolved by two-dimensional chromatography, visualized, and quantitated by phosphorimaging. The data represent the average of three independent experiments. The bulk incorporation of label into phsopholipid for these strains during the pulse was essentially equivalent (see Figure 3A), and the relative magnitudes of PtdIns-4-P were directly related to quantities of PtdIns-4-P per cell equivalent in these strains. (C) A set of isogenic sec14-1^ts, Δspo14 strains carrying the indicated bypass Sec14p mutations were streaked for single colonies on YPD medium and incubated for 72 h at 33.5°C. The corresponding sac1-26, pct1-2, kes1-1, and BSD1-124 derivatives were represented by strains CTY1127, CTY1096, CTY1098, and CTY1129, respectively. The SEC14, Δspo14 derivative (CTY1092) served as a positive growth control, whereas the sec14-1^ts, Δspo14 strain (CTY1079) represented the negative control. In the Δspo14 genetic background, neither sac1, kes1 nor BSD1-124 alleles imparted any detectable phenotypic suppression of sec14 growth defects. By contrast, inactivation of the CDP-choline pathway via the pct1-2 mutation (and cki1; our unpublished results) retained a clear PLD-independent ability to suppress sec14^ts growth defects under these conditions.

The inositol auxotrophy of Δsac1 strains is not the consequence of defects in INO1 expression. (A) The yeast strains with the indicated genotypes were streaked for isolation on uracil-deficient medium in the presence (+Inositol) or absence (−Inositol) of inositol and incubated at 26°C for 72 h. The YEp(P_SEC14:: INO1) plasmid, which drives constitutive expression of INO1 from the SEC14 promoter, was incapable of rescuing inositol prototrophy in the Δsac1 strain CTY244. That this plasmid effectively drove INO1 expression is amply demonstrated by its ability to rescue growth of the ino1-13 mutant (CTY) on inositol-free medium. (B) Wild-type (CTY182) and Δsac1 (CTY244) strains were grown in YPD medium to midlogarithmic growth phase, harvested, and washed. The cultures were split and either shifted to minimal medium containing both inositol and choline (+Ino) or to inositol- and choline-free medium (−Ino). After 4 h of incubation at 30°C with shaking, cells were harvested, and total RNA was recovered and subjected to Northern analysis using specific probes for INO1 and OPI3 mRNA that were generated by random priming. Each lane was loaded with 10 μg of RNA. The results clearly demonstrate that both INO1 and OPI3 expression was induced in cells shifted to inositol- and choline-free medium. To confirm proper normalization of RNA load, these same RNA samples were also probed for messages (e.g., PSS1) that are present under both the Ino⁻ and Ino⁺ conditions. As expected, PSS1 mRNA was detected in all lanes (our unpublished results).

DGK expression suppresses the inositol auxotrophy associated with Δsac1 strains. (A) The indicated yeast strains were streaked for isolation on solid uracil-deficient minimal medium that either contained inositol (+Inositol) or was inositol free (−Inositol). Plates were incubated at 26°C for 72 h. Wild-type and Δsac1 strains were CTY182 and CTY244, respectively. Episomal DGK expression plasmids and cognate vector-only controls are designated YEpDGK and YEpURA3, respectively. (B) Mutations that block PtdCho biosynthesis by the CDP-choline pathway suppress the inositol auxotrophy of Δsac1 strains. Yeast strains with the indicated genotypes were streaked for isolation on solid minimal defined medium that was either supplemented with inositol (+Inositol) or was inositol-free (−Inositol). Plates were incubated at 26°C for 72 h and scored for growth. Wild-type and Δsac1 strains were CTY182 and CTY244, respectively. The Δcki1 and Δopi3 derivatives were generated by transformation of yeast cells with the appropriate disruption constructs and confimed by genomic Southern analysis. In these experiments, heavy inocula of the Δsac1 and Δsac1, Δopi3 strains were streaked onto the −Inositol plates to illustrate the tightness of the inositol auxotrophy.

Effects of inositol starvation on cell viability. Wild-type (CTY182; closed circles), Δsac1 (CTY244; closed squares), Δsac1, Δcki1 (CTY; stippled cicles), and ino1-13 (CTY; open squares) strains were grown in minimal defined medium supplemented with inositol, harvested, washed, and shifted to inositol-free medium at time zero. Samples of each culture were subsequently removed at the indicated times after shift and serially diluted in YPD medium, and the serial dilutions were immediately plated onto YPD agar plates. Individual colonies were scored after 3 d of growth at 26°C to assess viable cell counts. Data are from a representative experiment.

Bypass Sec14p mechanisms. The relevant lipid metabolic pathways and proteins that determine the activity of these pathways are shown. The key lipids (i.e., PtdIns-4-P, PtdCho, and DAG) are indicated in bold. Key proteins and their corresponding sites of action are indicated in the black boxes. We propose two principal mechanisms for bypass Sec14p. First, we suggest that Kes1p, SacIp, and Bsd1p are involved in a PLD regulatory pathway that, in yeast mutants carrying the corresponding bypass Sec14p alleles, results in PLD activation and subsequent production of DAG. This particular pathway recapitulates the physiological function for the PtdIns-bound form of Sec14p (Sec14p∼PI) that we have proposed effects DAG production via regulation of inositol phospholipid metabolism (Kearns et al., 1997). Loss of SacIp function results in PtdIns-4-P accumulation that, in turn, either activates Bsd1p (identified by dominant bypass Sec14p alleles; Cleves et al., 1991b; Fang et al., 1996) or inactivates Kes1p (identified by recessive bypass Sec14p alleles; Fang et al., 1996). The consequence is activation of PLD, which results in DAG production through the metabolism of the PtdOH generated by PLD-mediated hydrolysis of PtdCho. We propose that the excess DAG generated via this route is consumed by the CDP-choline pathway for PtdCho biosynthesis, thereby resulting in the observed activation of this pathway biosynthetic pathway in sac1 mutants. Second, we suggest that dysfunction of any one of the enzymes of the CDP-choline pathway results in the observed recessive bypass Sec14p phenotypes (Cleves et al., 1991b), because inactivation of the CDP-choline pathway recapitulates the function of PtdCho-bound Sec14p (Sec14p∼PC) (Skinner et al., 1995). Although bypass Sec14p via this route also requires PLD activity, there is nonetheless a measurable PLD-independent component to suppression of Sec14p defects by CDP-choline pathway defects.