Figure 5.

Gcs1p and Age2p act downstream of PLD in the Sec14p pathway. PLD activation in Δgcs1and Δage2 yeast strains as measured by PA production. Strains carrying sec14-1^ts are identified by the bar at top. Acute activation of PLD in sec14-1^ts strains is evoked by shifting cells from 26 to 33.5°C, but this shift has no effect on the PLD activity of wild-type (WT) yeast strains (Xie et al., 1998). The positive PLD activation control is a sec14-1^ts strain carrying a bypass Sec14p allele (pct1-2), whereas the negative control is an isogenic PLD-deficient (Δspo14) derivative (relevant genotypes at bottom). The isogenic Δgcs1 and Δage2 derivatives are identified at bottom. Yeast strains were labeled to steady state with [³²P]orthophosphate in minimal medium at 26°C. Cells were harvested, washed, resuspended in fresh label-free medium, and incubated at 33.5°C for 3 h to induce Sec14p deficiency. Phospholipids were extracted, resolved by two-dimensional thin-layer chromatography, and quantified by phosphorimaging. Values are expressed as (³²P in PA/total ³²P in extractable phospholipid) × 100% (n ≥ 3). All strains equally incorporated ³²P into phospholipid per unit cell number.