FIGURE 2.

Identification of ScGPCAT (GPC1). Yeast extracts of strains from the yeast deletion collection were incubated with [¹⁴C]GPC and 18:1-CoA. [¹⁴C]G3P was added to the incubation as an internal control of enzymatic activities. A, enzyme reactions in yeast from the substrates [¹⁴C]GPC and [¹⁴C]G3P. The yeast-acylating enzymes are indicated. B, autoradiogram of the screening on thin layer chromatography plates showing the deletion strain lacking GPCAT activity (within the hatched square). C, complementation of YGR149W (GPC1) in the gpc1Δ deletion strain. Empty vector as well as the deletion strain (gpc1Δ) are also included. Irrelevant lanes from the plate have been removed from the picture (marked by a vertical line). D, Western blot of Gpc1p. Wild type strain bearing EV or a plasmid (GPC1-V5) harboring GPC1 under the control of the galactose-inducible GAL1 promoter and containing a C-terminal V5 epitope. Cells were grown on either glucose or galactose. Equivalent amounts of protein (75 μg) were loaded onto each lane. Anti-Gpc1p-V5 primary antibody and goat anti-mouse secondary antibody were employed. The blot was visualized using an Odyssey FC imaging system. G6PD was used as the loading control.