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. 1999 Jul;10(7):2235–2250. doi: 10.1091/mbc.10.7.2235

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Copyright © 1999, The American Society for Cell Biology

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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 By contrast, inactivation of the CDP-choline pathway via the pct1-2 mutation (and cki1; our unpublished results) retained a clear PLD-independent ability to suppress sec14^ts growth defects under these conditions.

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.