Fig. 2

Stt4 PIK patches localize to ER-PM contact sites. a Schematic representation of two Stt4 PI 4-kinase complexes present at the PM. Stt4 complex I (Stt4/Efr3/Ypp1), also known as a PIK patch, is enriched in mother cells and is essential for Osh3 PM localization. ORP/Osh protein family members are proposed to mediate lipid transfer and exchange at ER-PM contacts resulting in PI4P consumption by the Sac1 phosphatase. Complex II (Stt4/Efr3/Sfk1) is involved in stimulus-induced PI4P and PI(4,5)P₂ synthesis. b Sfk1 (Stt4 Complex II) is not required for heat-induced PI4P signaling in mother cells. GFP-P4C fluorescence indicating PI4P localization in wild type (left panels) and sfk1Δ cells (right panels) at 26 °C and 42 °C. Arrows point out increased PI4P levels at the PM of mother cells. Scale bar, 5 μm. c Stt4 complex I (PIK patches) are ER-associated. Wild type cells expressing GFP-Stt4 or Ypp1-GFP (green) co-expressed the ER marker DsRed-HDEL (magenta). Mother (m) and daughter (d) cells are indicated (dashed). Stt4- and Ypp1-containing PIK patches (outlined in white) were automatically segmented and scored for co-localization with the ER marker. Scale bar, 4 μm. d High-content quantitative analysis of PIK patch localization with the ER. Maxima from GFP-Stt4 puncta (7264 from 1111 cells) and Ypp1-GFP puncta (13,079 from 1527 cells) were identified using Fiji. Maxima positive for the ER marker DsRed-HDEL were scored as ER-associated. Results show the mean and standard deviation from three independent experiments