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. 2002 Apr 1;157(1):63–78. doi: 10.1083/jcb.200201037

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Copyright © 2002, The Rockefeller University Press

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Figure 8. — Kes1p dosage and “bypass Sec14p”. (A) Distinct sensitivities of different mechanisms for “bypass Sec14p” to increased Kes1p dosages. Kes1p was modestly overexpressed by introduction of YCp (KES1), or highly overexpressed by introduction of YEp(PPGK:: KES1), which drives Kes1p expression from a powerful PGK promoter (see Materials and methods). All mechanisms of “bypass Sec14p” except that mediated by sac1 are sensitive to modest increases (two- to threefold) in Kes1p levels. A high degree (∼20-fold) of Kes1p is required for compromise of “bypass Sec14p” in sac1 mutants. (B) Structure of Sac1pΔTM. The first 462 codons of Sac1p, which include the catalytic domain but not the transmembrane domain, were fused to a protein A tag. This construct is designated Sac1pΔTM and its expression is under control of the native SAC1 promoter. Details of the construction are available from the authors by request. (C) PI(4)P levels as a function of Sac1pΔTM expression. A wild-type SAC1 strain, a sac1 mutant strain, and isogenic sac1 derivatives transformed either with YCp(sac1 ^Δ ^TM) or YEp(sac1 ^Δ ^TM) were pulse radiolabeled for 20 min at 26°C with [³²P]-orthophosphate (10 μCi/ml), phospholipids were extracted under acidic conditions, lipids were resolved by two-dimensional paper chromatography, and PI(4)P was identified and quantified by phosphorimaging (Rivas et al., 1999). PI(4)P is expressed as a percentage of total extractable phospholipid. Relevant strain genotypes are indicated. The various strains incorporated similar amounts of [³²P]-radiolabel into total lipid. Data are expressed as the mean ± standard deviation from at least three independent experiments. (D) Sac1pΔTM expression sensitizes sac1-mediated “bypass Sec14p” to Kes1p. Isogenic sac1 and sac1/YEp(sac1 ^Δ ^TM) yeast strains were transformed with YCp(KES1). Transformants were subsequently isolated and streaked for isolated colonies on selective medium at 37°C. Growth was scored after 48 h of incubation. Relevant genotypes are given.

Figure 8. — Kes1p dosage and “bypass Sec14p”. (A) Distinct sensitivities of different mechanisms for “bypass Sec14p” to increased Kes1p dosages. Kes1p was modestly overexpressed by introduction of YCp (KES1), or highly overexpressed by introduction of YEp(PPGK:: KES1), which drives Kes1p expression from a powerful PGK promoter (see Materials and methods). All mechanisms of “bypass Sec14p” except that mediated by sac1 are sensitive to modest increases (two- to threefold) in Kes1p levels. A high degree (∼20-fold) of Kes1p is required for compromise of “bypass Sec14p” in sac1 mutants. (B) Structure of Sac1pΔTM. The first 462 codons of Sac1p, which include the catalytic domain but not the transmembrane domain, were fused to a protein A tag. This construct is designated Sac1pΔTM and its expression is under control of the native SAC1 promoter. Details of the construction are available from the authors by request. (C) PI(4)P levels as a function of Sac1pΔTM expression. A wild-type SAC1 strain, a sac1 mutant strain, and isogenic sac1 derivatives transformed either with YCp(sac1 ^Δ ^TM) or YEp(sac1 ^Δ ^TM) were pulse radiolabeled for 20 min at 26°C with [³²P]-orthophosphate (10 μCi/ml), phospholipids were extracted under acidic conditions, lipids were resolved by two-dimensional paper chromatography, and PI(4)P was identified and quantified by phosphorimaging (Rivas et al., 1999). PI(4)P is expressed as a percentage of total extractable phospholipid. Relevant strain genotypes are indicated. The various strains incorporated similar amounts of [³²P]-radiolabel into total lipid. Data are expressed as the mean ± standard deviation from at least three independent experiments. (D) Sac1pΔTM expression sensitizes sac1-mediated “bypass Sec14p” to Kes1p. Isogenic sac1 and sac1/YEp(sac1 ^Δ ^TM) yeast strains were transformed with YCp(KES1). Transformants were subsequently isolated and streaked for isolated colonies on selective medium at 37°C. Growth was scored after 48 h of incubation. Relevant genotypes are given.