Evidence for an intrinsic toxicity of phosphatidylcholine to Sec14p-dependent protein transport from the yeast Golgi complex

Abstract

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient "bypass Sec14p" phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1(ts)-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.

Figure 1

(A) The two pathways for PtdCho biosynthesis in yeast. Mutations in CDP-choline pathway, but not in PtdEtn-methylation pathway, suppress the essential requirement of Sec14p for Golgi secretory function and cell viability. CDP-choline pathway activity is supported by endogenous choline derived from PtdCho turnover, and by exogenous choline, which is captured by the choline transporter. In the absence of exogenous choline, PtdCho turnover represents the sole pathway for choline production. Genetic designations for the structural genes encoding enzymes for PtdCho synthesis and turnover are given at the corresponding execution points. The role of the choline transporter is also illustrated. (B) Choline-sensitive suppressors of sec14 (css). Indicated strains were patched on YPD plate and incubated at 26°C for 24 h. The cell patches were then replica plated onto minimal plates supplemented with 1 mM inositol but no choline (I⁺C⁻), or with 1 mM inositol and choline at indicated concentrations (I⁺ 5 μM Cho and I⁺ 10 μM Cho), or neither inositol nor choline (I⁻C⁻). The replica plates were then incubated at 37°C for 48 h. Isogenic strains were used: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts css1), CTY1375 (sec14-1^ts css2), CTY1473 (sec14-1^ts css3).

Figure 1

(A) The two pathways for PtdCho biosynthesis in yeast. Mutations in CDP-choline pathway, but not in PtdEtn-methylation pathway, suppress the essential requirement of Sec14p for Golgi secretory function and cell viability. CDP-choline pathway activity is supported by endogenous choline derived from PtdCho turnover, and by exogenous choline, which is captured by the choline transporter. In the absence of exogenous choline, PtdCho turnover represents the sole pathway for choline production. Genetic designations for the structural genes encoding enzymes for PtdCho synthesis and turnover are given at the corresponding execution points. The role of the choline transporter is also illustrated. (B) Choline-sensitive suppressors of sec14 (css). Indicated strains were patched on YPD plate and incubated at 26°C for 24 h. The cell patches were then replica plated onto minimal plates supplemented with 1 mM inositol but no choline (I⁺C⁻), or with 1 mM inositol and choline at indicated concentrations (I⁺ 5 μM Cho and I⁺ 10 μM Cho), or neither inositol nor choline (I⁻C⁻). The replica plates were then incubated at 37°C for 48 h. Isogenic strains were used: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts css1), CTY1375 (sec14-1^ts css2), CTY1473 (sec14-1^ts css3).

Figure 2

Steady-state phospholipid profiles of css mutants in defined I⁺C⁺ media. Cells were grown for five to six generations at 26°C in I⁺C⁺ (A) or I⁺C⁻ media (B) supplemented with [³²P]orthophosphate to 10 μCi/ml. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphorimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. Strains used were isogenic and included: CTY1-1A (sec14-1^ts: black bars), CTY1374 (sec14-1^ts css1; hatched bars), CTY1375 (sec14-1^ts css2, stippled bars), CTY1473 (sec14-1^ts css3; open bars). These data represent the averages of three independent experiments. PtdSer, phosphatidylserine.

Figure 2

Steady-state phospholipid profiles of css mutants in defined I⁺C⁺ media. Cells were grown for five to six generations at 26°C in I⁺C⁺ (A) or I⁺C⁻ media (B) supplemented with [³²P]orthophosphate to 10 μCi/ml. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphorimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. Strains used were isogenic and included: CTY1-1A (sec14-1^ts: black bars), CTY1374 (sec14-1^ts css1; hatched bars), CTY1375 (sec14-1^ts css2, stippled bars), CTY1473 (sec14-1^ts css3; open bars). These data represent the averages of three independent experiments. PtdSer, phosphatidylserine.

Figure 3

(A) Prevention of choline uptake enables PtdEtn-methylation pathway dysfunction to efficiently suppress sec14-1^ts growth defects. Appropriate strains were streaked on indicated plates and incubated at 37°C for 48 h. Strains used were: CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3), CTY1498 (hnm1Δ::URA3), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3), CTY1501 (sec14-1^ts opi3 hnm1Δ::URA3). (B) PtdEtn-methylation pathway mutations efficiently restore growth to the sec14-1^ts strain at 37°C in liquid I⁺C⁻ media. Appropriate strains were picked up from freshly streaked YPD plates, inoculated into liquid defined media supplemented with inositol but no choline, and incubated at 37°C with shaking. Aliquots were collected for determination of OD₆₀₀ at indicated times. Strains used were CTY182 (wild-type; closed diamonds), CTY1-1A (sec14-1^ts; closed squares), CTY1375 (sec14-1^ts opi3; closed triangles), CTY1377 (sec14-1^ts opi3 hnm1Δ::URA3; open triangles). (C) Choline recapture visualized by cross-feeding. Patches of a sec14-1^ts opi3 strain and a sec14-1^ts opi3 hnm1Δ strain (choline feeders) were deposited on a lawn of sec14-1^ts opi3 strain (indicator) on an I⁺C⁻ plate and incubated at 37°C for 36 h. The growth of indicator cells surrounding the sec14-1^ts opi3 hnm1Δ feeder was inhibited, as indicated by a halo surrounding the feeder. This inhibition did not occur when the sec14-1^ts opi3 strain, which is competent for choline reuptake, was used as feeder. No halo was observed when the incubation temperature is 26°C or when the sec14-1^ts opi3 hnm1Δ strain was used as indicator. Strains used were isogenic and included: CTY1375 (sec14-1^ts opi3), CTY1500 (sec14-1^ts opi3 hnm1Δ::URA3). (D) Rate of PtdCho biosynthesis via CDP-choline pathway correlates with the suppression of sec14 growth defects in I⁺C⁺ or I⁺C⁻ media. Indicated strains were cultured to early logarithmic phase at 26°C in either I⁺C⁺ (black bars) or I⁺C⁻ minimal media (open bars). Cultures were subsequently shifted to 33.5°C for 1 h, and pulse-radiolabeled with [³²P]orthophosphate (10 μCi/ml) at 33.5°C for 20 min. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphoimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. The strains used were isogenic and included: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY102 (sec14-1^ts pct1-2), CTY1374 (sec14-1^ts cho2), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3). These data represent averages from at least three independent experiments.

Figure 3

(A) Prevention of choline uptake enables PtdEtn-methylation pathway dysfunction to efficiently suppress sec14-1^ts growth defects. Appropriate strains were streaked on indicated plates and incubated at 37°C for 48 h. Strains used were: CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3), CTY1498 (hnm1Δ::URA3), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3), CTY1501 (sec14-1^ts opi3 hnm1Δ::URA3). (B) PtdEtn-methylation pathway mutations efficiently restore growth to the sec14-1^ts strain at 37°C in liquid I⁺C⁻ media. Appropriate strains were picked up from freshly streaked YPD plates, inoculated into liquid defined media supplemented with inositol but no choline, and incubated at 37°C with shaking. Aliquots were collected for determination of OD₆₀₀ at indicated times. Strains used were CTY182 (wild-type; closed diamonds), CTY1-1A (sec14-1^ts; closed squares), CTY1375 (sec14-1^ts opi3; closed triangles), CTY1377 (sec14-1^ts opi3 hnm1Δ::URA3; open triangles). (C) Choline recapture visualized by cross-feeding. Patches of a sec14-1^ts opi3 strain and a sec14-1^ts opi3 hnm1Δ strain (choline feeders) were deposited on a lawn of sec14-1^ts opi3 strain (indicator) on an I⁺C⁻ plate and incubated at 37°C for 36 h. The growth of indicator cells surrounding the sec14-1^ts opi3 hnm1Δ feeder was inhibited, as indicated by a halo surrounding the feeder. This inhibition did not occur when the sec14-1^ts opi3 strain, which is competent for choline reuptake, was used as feeder. No halo was observed when the incubation temperature is 26°C or when the sec14-1^ts opi3 hnm1Δ strain was used as indicator. Strains used were isogenic and included: CTY1375 (sec14-1^ts opi3), CTY1500 (sec14-1^ts opi3 hnm1Δ::URA3). (D) Rate of PtdCho biosynthesis via CDP-choline pathway correlates with the suppression of sec14 growth defects in I⁺C⁺ or I⁺C⁻ media. Indicated strains were cultured to early logarithmic phase at 26°C in either I⁺C⁺ (black bars) or I⁺C⁻ minimal media (open bars). Cultures were subsequently shifted to 33.5°C for 1 h, and pulse-radiolabeled with [³²P]orthophosphate (10 μCi/ml) at 33.5°C for 20 min. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphoimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. The strains used were isogenic and included: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY102 (sec14-1^ts pct1-2), CTY1374 (sec14-1^ts cho2), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3). These data represent averages from at least three independent experiments.

Figure 3

(A) Prevention of choline uptake enables PtdEtn-methylation pathway dysfunction to efficiently suppress sec14-1^ts growth defects. Appropriate strains were streaked on indicated plates and incubated at 37°C for 48 h. Strains used were: CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3), CTY1498 (hnm1Δ::URA3), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3), CTY1501 (sec14-1^ts opi3 hnm1Δ::URA3). (B) PtdEtn-methylation pathway mutations efficiently restore growth to the sec14-1^ts strain at 37°C in liquid I⁺C⁻ media. Appropriate strains were picked up from freshly streaked YPD plates, inoculated into liquid defined media supplemented with inositol but no choline, and incubated at 37°C with shaking. Aliquots were collected for determination of OD₆₀₀ at indicated times. Strains used were CTY182 (wild-type; closed diamonds), CTY1-1A (sec14-1^ts; closed squares), CTY1375 (sec14-1^ts opi3; closed triangles), CTY1377 (sec14-1^ts opi3 hnm1Δ::URA3; open triangles). (C) Choline recapture visualized by cross-feeding. Patches of a sec14-1^ts opi3 strain and a sec14-1^ts opi3 hnm1Δ strain (choline feeders) were deposited on a lawn of sec14-1^ts opi3 strain (indicator) on an I⁺C⁻ plate and incubated at 37°C for 36 h. The growth of indicator cells surrounding the sec14-1^ts opi3 hnm1Δ feeder was inhibited, as indicated by a halo surrounding the feeder. This inhibition did not occur when the sec14-1^ts opi3 strain, which is competent for choline reuptake, was used as feeder. No halo was observed when the incubation temperature is 26°C or when the sec14-1^ts opi3 hnm1Δ strain was used as indicator. Strains used were isogenic and included: CTY1375 (sec14-1^ts opi3), CTY1500 (sec14-1^ts opi3 hnm1Δ::URA3). (D) Rate of PtdCho biosynthesis via CDP-choline pathway correlates with the suppression of sec14 growth defects in I⁺C⁺ or I⁺C⁻ media. Indicated strains were cultured to early logarithmic phase at 26°C in either I⁺C⁺ (black bars) or I⁺C⁻ minimal media (open bars). Cultures were subsequently shifted to 33.5°C for 1 h, and pulse-radiolabeled with [³²P]orthophosphate (10 μCi/ml) at 33.5°C for 20 min. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphoimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. The strains used were isogenic and included: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY102 (sec14-1^ts pct1-2), CTY1374 (sec14-1^ts cho2), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3). These data represent averages from at least three independent experiments.

Figure 3

(A) Prevention of choline uptake enables PtdEtn-methylation pathway dysfunction to efficiently suppress sec14-1^ts growth defects. Appropriate strains were streaked on indicated plates and incubated at 37°C for 48 h. Strains used were: CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3), CTY1498 (hnm1Δ::URA3), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3), CTY1501 (sec14-1^ts opi3 hnm1Δ::URA3). (B) PtdEtn-methylation pathway mutations efficiently restore growth to the sec14-1^ts strain at 37°C in liquid I⁺C⁻ media. Appropriate strains were picked up from freshly streaked YPD plates, inoculated into liquid defined media supplemented with inositol but no choline, and incubated at 37°C with shaking. Aliquots were collected for determination of OD₆₀₀ at indicated times. Strains used were CTY182 (wild-type; closed diamonds), CTY1-1A (sec14-1^ts; closed squares), CTY1375 (sec14-1^ts opi3; closed triangles), CTY1377 (sec14-1^ts opi3 hnm1Δ::URA3; open triangles). (C) Choline recapture visualized by cross-feeding. Patches of a sec14-1^ts opi3 strain and a sec14-1^ts opi3 hnm1Δ strain (choline feeders) were deposited on a lawn of sec14-1^ts opi3 strain (indicator) on an I⁺C⁻ plate and incubated at 37°C for 36 h. The growth of indicator cells surrounding the sec14-1^ts opi3 hnm1Δ feeder was inhibited, as indicated by a halo surrounding the feeder. This inhibition did not occur when the sec14-1^ts opi3 strain, which is competent for choline reuptake, was used as feeder. No halo was observed when the incubation temperature is 26°C or when the sec14-1^ts opi3 hnm1Δ strain was used as indicator. Strains used were isogenic and included: CTY1375 (sec14-1^ts opi3), CTY1500 (sec14-1^ts opi3 hnm1Δ::URA3). (D) Rate of PtdCho biosynthesis via CDP-choline pathway correlates with the suppression of sec14 growth defects in I⁺C⁺ or I⁺C⁻ media. Indicated strains were cultured to early logarithmic phase at 26°C in either I⁺C⁺ (black bars) or I⁺C⁻ minimal media (open bars). Cultures were subsequently shifted to 33.5°C for 1 h, and pulse-radiolabeled with [³²P]orthophosphate (10 μCi/ml) at 33.5°C for 20 min. Bulk glycerophospholipids were extracted, resolved, and quantitated by phosphoimaging. Incorporation of ³²P into each phospholipid species is indicated as a percentage of total incorporation of radiolabel into extractable phospholipid. The strains used were isogenic and included: CTY182 (wild-type), CTY1-1A (sec14-1^ts), CTY102 (sec14-1^ts pct1-2), CTY1374 (sec14-1^ts cho2), CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1500 (sec14-1^ts cho2 hnm1Δ::URA3). These data represent averages from at least three independent experiments.

Figure 4

(A) Efficiency of invertase secretion at 37°C for PtdEtn-methylation pathway mutants grown in I⁺C⁺ (black bars) and I⁺C⁻ media (open bars). Secretion index was calculated as (extracellular invertase/total invertase × 100%) (Salama et al., 1990), and the values presented represent the averages of triplicate determinations from at least three independent experiments. The secretion indices of wild-type strain and sec14-1^ts strain represent the secretory efficiency under Sec14p-proficient and Sec14p-deficient conditions, respectively. Strains used were CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3). (B) Trafficking of CPY through the secretory pathway to vacuole in I⁺C⁺ and I⁺C⁻ media. Appropriate strains were grown at 26°C to early logarithmic phase in the indicated media (I⁺C⁺ media are indicated by “+” and I⁺C⁻ media was indicated by “−” below the lane), shifted to 37°C for 2 h, and pulse-radiolabeled with ³⁵S-amino acids at 37°C for 30 min. Radiolabeled CPY species were recovered, resolved, and quantitated as described (Fang et al., 1996). The p1 (ER), p2 (Golgi), and mature vacuolar (m) forms of CPY are indicated at right. Strains used included CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), and CTY1375 (sec14-1^ts opi3).

Figure 4

(A) Efficiency of invertase secretion at 37°C for PtdEtn-methylation pathway mutants grown in I⁺C⁺ (black bars) and I⁺C⁻ media (open bars). Secretion index was calculated as (extracellular invertase/total invertase × 100%) (Salama et al., 1990), and the values presented represent the averages of triplicate determinations from at least three independent experiments. The secretion indices of wild-type strain and sec14-1^ts strain represent the secretory efficiency under Sec14p-proficient and Sec14p-deficient conditions, respectively. Strains used were CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), CTY1375 (sec14-1^ts opi3). (B) Trafficking of CPY through the secretory pathway to vacuole in I⁺C⁺ and I⁺C⁻ media. Appropriate strains were grown at 26°C to early logarithmic phase in the indicated media (I⁺C⁺ media are indicated by “+” and I⁺C⁻ media was indicated by “−” below the lane), shifted to 37°C for 2 h, and pulse-radiolabeled with ³⁵S-amino acids at 37°C for 30 min. Radiolabeled CPY species were recovered, resolved, and quantitated as described (Fang et al., 1996). The p1 (ER), p2 (Golgi), and mature vacuolar (m) forms of CPY are indicated at right. Strains used included CTY182 (wild type), CTY1-1A (sec14-1^ts), CTY1374 (sec14-1^ts cho2), and CTY1375 (sec14-1^ts opi3).

Figure 5

Role of phospholipase D in suppression of sec14 defects by PtdEtn-methylation pathway dysfunction. (A) Role of PLD (SPO14 gene product). The indicated strains were streaked onto YPD plates, incubated at 37°C for 48 h, and the growth of each strain was then recorded. Isogenic strains were used and these included CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1502 (hnm1Δ::HIS3), CTY1504 (sec14-1^ts cho2 hnm1Δ::HIS3), CTY1505 (sec14-1^ts opi3 hnm1Δ::HIS3), CTY1506 (hnm1Δ::HIS3 spo14Δ::URA3), CTY1507 (sec14-1^ts hnm1Δ::URA3 spo14Δ::HIS3), CTY1512 (sec14-1^ts cho2 hnm1Δ::HIS3 spo14Δ::URA3), CTY1513 (sec14-1^ts opi3 hnm1Δ::HIS3 spo14Δ::URA3). (B) The catalytic activity of PLD is necessary, but not sufficient, for the suppression of sec14 defects by PtdEtn-methylation pathway defects. The sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3 strain and its derivative strains carrying either a wild-type SPO14 gene on low copy plasmid (YCp), or a mutant spo14 gene on low copy plasmid (YCp) or high copy plasmid (YEp), were streaked on YPD plates and incubated at 37°C for 48 h. The growth of each strain was then recorded. Strains used included CTY1485 (sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3); CTY1519 (CTY1485/YCpSPO14); CTY1520 (CTY1485/YCp spo14^K→H); CTY1521 (CTY1485/YCp spo14^ΔN); CTY1522 (CTY1485/YEp spo14^K→H); CTY1523 (CTY1485/YEp spo14^ΔN). (C) Plb1p overexpression improves growth of sec14-1^ts mutants at a nonpermissive temperature. Isogenic yeast strains CTY182 (SEC14) and CTY1–1A (sec14-1^ts) were transformed with the indicated YEp plasmids. Transformants were isolated and streaked for isolated colonies on YPD medium at 35°C. Growth was scored after 48 h of incubation. Relevant genotypes are shown. The SEC14/YEp(URA3) and sec14-1^ts/YEp(URA3) strains represented positive and negative controls for growth, respectively.

Figure 5

Role of phospholipase D in suppression of sec14 defects by PtdEtn-methylation pathway dysfunction. (A) Role of PLD (SPO14 gene product). The indicated strains were streaked onto YPD plates, incubated at 37°C for 48 h, and the growth of each strain was then recorded. Isogenic strains were used and these included CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1502 (hnm1Δ::HIS3), CTY1504 (sec14-1^ts cho2 hnm1Δ::HIS3), CTY1505 (sec14-1^ts opi3 hnm1Δ::HIS3), CTY1506 (hnm1Δ::HIS3 spo14Δ::URA3), CTY1507 (sec14-1^ts hnm1Δ::URA3 spo14Δ::HIS3), CTY1512 (sec14-1^ts cho2 hnm1Δ::HIS3 spo14Δ::URA3), CTY1513 (sec14-1^ts opi3 hnm1Δ::HIS3 spo14Δ::URA3). (B) The catalytic activity of PLD is necessary, but not sufficient, for the suppression of sec14 defects by PtdEtn-methylation pathway defects. The sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3 strain and its derivative strains carrying either a wild-type SPO14 gene on low copy plasmid (YCp), or a mutant spo14 gene on low copy plasmid (YCp) or high copy plasmid (YEp), were streaked on YPD plates and incubated at 37°C for 48 h. The growth of each strain was then recorded. Strains used included CTY1485 (sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3); CTY1519 (CTY1485/YCpSPO14); CTY1520 (CTY1485/YCp spo14^K→H); CTY1521 (CTY1485/YCp spo14^ΔN); CTY1522 (CTY1485/YEp spo14^K→H); CTY1523 (CTY1485/YEp spo14^ΔN). (C) Plb1p overexpression improves growth of sec14-1^ts mutants at a nonpermissive temperature. Isogenic yeast strains CTY182 (SEC14) and CTY1–1A (sec14-1^ts) were transformed with the indicated YEp plasmids. Transformants were isolated and streaked for isolated colonies on YPD medium at 35°C. Growth was scored after 48 h of incubation. Relevant genotypes are shown. The SEC14/YEp(URA3) and sec14-1^ts/YEp(URA3) strains represented positive and negative controls for growth, respectively.

Figure 5

Role of phospholipase D in suppression of sec14 defects by PtdEtn-methylation pathway dysfunction. (A) Role of PLD (SPO14 gene product). The indicated strains were streaked onto YPD plates, incubated at 37°C for 48 h, and the growth of each strain was then recorded. Isogenic strains were used and these included CTY1499 (sec14-1^ts hnm1Δ::URA3), CTY1502 (hnm1Δ::HIS3), CTY1504 (sec14-1^ts cho2 hnm1Δ::HIS3), CTY1505 (sec14-1^ts opi3 hnm1Δ::HIS3), CTY1506 (hnm1Δ::HIS3 spo14Δ::URA3), CTY1507 (sec14-1^ts hnm1Δ::URA3 spo14Δ::HIS3), CTY1512 (sec14-1^ts cho2 hnm1Δ::HIS3 spo14Δ::URA3), CTY1513 (sec14-1^ts opi3 hnm1Δ::HIS3 spo14Δ::URA3). (B) The catalytic activity of PLD is necessary, but not sufficient, for the suppression of sec14 defects by PtdEtn-methylation pathway defects. The sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3 strain and its derivative strains carrying either a wild-type SPO14 gene on low copy plasmid (YCp), or a mutant spo14 gene on low copy plasmid (YCp) or high copy plasmid (YEp), were streaked on YPD plates and incubated at 37°C for 48 h. The growth of each strain was then recorded. Strains used included CTY1485 (sec14-1^ts opi3 hnm1Δ::URA3 spo14Δ::URA3); CTY1519 (CTY1485/YCpSPO14); CTY1520 (CTY1485/YCp spo14^K→H); CTY1521 (CTY1485/YCp spo14^ΔN); CTY1522 (CTY1485/YEp spo14^K→H); CTY1523 (CTY1485/YEp spo14^ΔN). (C) Plb1p overexpression improves growth of sec14-1^ts mutants at a nonpermissive temperature. Isogenic yeast strains CTY182 (SEC14) and CTY1–1A (sec14-1^ts) were transformed with the indicated YEp plasmids. Transformants were isolated and streaked for isolated colonies on YPD medium at 35°C. Growth was scored after 48 h of incubation. Relevant genotypes are shown. The SEC14/YEp(URA3) and sec14-1^ts/YEp(URA3) strains represented positive and negative controls for growth, respectively.

Figure 6

PtdCho toxicity for Sec14p-dependent Golgi secretory function. We propose PtdCho is toxic to Sec14p-dependent Golgi secretory function because of its negative effects on the activities of positive downstream effectors. Defects in PtdCho biosynthesis rescue viability of Sec14p-deficient cells when the CDP-choline pathway for PtdCho biosynthesis is inactivated, or is operating at low levels, and PLD is activated. This condition is a function of the efficiency with which the pool of liberated choline that is excreted from cells is recaptured by the choline transporter (Hnm1p). The recaptured choline is metabolically channeled into the CDP-choline pathway. PtdOH and DAG are also produced by PLD; in the former case directly, and in the latter case by PtdOH-phosphohydrolase (PAP)-mediated dephosphorylation of PtdOH. We propose that the combinatorial regulation of PtdCho, DAG, and PtdOH metabolism by Sec14p (or PLD under conditions of Sec14p dysfunction) is required for at least one essential pathway for protein transport from the yeast Golgi complex.