Figure 3.

Pah1 regulates PM–ER contacts. (A,C) Serial dilutions of (A) Δscs2Δice2 and (C) Δscs2Δice2Δpct1 yeast overexpressing GST–Pah1 from a plasmid under control of the galactose promoter (+pGST–PAH1) on SD media containing either glucose (Dex) or galactose (Gal). (B) Yeast expressing either HA–Pah1 or the catalytically inactive D398E mutant from a plasmid grown on SD media lacking choline. (D) Opi3 methylation assay for wild-type and Δscs2Δice2 cells overexpressing GST–Pah1. (E) Images of Tcb3-GFP in Δscs2Δice2 yeast overexpressing GST–Pah1. Scale bar, 2 μm. (F) Schematic of ultrastructural assay and representative transmission electron microscopy images illustrating PM–ER contacts (arrows). (G) Ratio of PM–ER contacts to PM perimeter. **P<10⁻⁴ versus WT; *P<0.001 versus WT; ##P<0.01 versus Δscs2Δice2, #P<0.05 versus Δscs2Δice2. (H) PM–ER contact length. *P<0.005 versus WT; #P<0.005 versus Δscs2Δice2. (I) Frequency of PM–ER contacts. **P<0.005 versus WT; *P<0.05 versus WT; ##P<0.005 versus Δscs2Δice2; #P<0.05 versus Δscs2Δice2. Error bars, s.e.m. ER, endoplasmic reticulum; GFP, green fluorescent protein; NS, not significant; PM, plasma membrane; SD, synthetic-defined; synth, synthesis; WT, wild-type.