ORP10 has an essential role, while OSBP and ORP9 play a redundant role in establishing/maintaining the ERTGoCS. (A) Graphical representation of the candidate proteins analyzed in the siRNA-based screen. The candidates possess a Golgi targeting motif represented by a PI4P-binding PH domain and an ER targeting motif represented by a FFAT motif (OSBP, ORP9, and CERT; Murphy and Levine, 2016) or a putative FFAT-like motif (FAPP2 [Mikitova and Levine, 2012] and ORP10 and ORP11 [Murphy and Levine, 2016]) that mediates the interaction with the VAP ER membrane proteins. FAPP1 possesses two potential FFAT motifs: 165 TFITTLE 171 (if phosphorylated on T165 and T168) and 242 TYSDTDS 248 (if phosphorylated on T242 and S248). (B) FLIM images of HeLa cells expressing TGN46-GFP and mCherry-Cb5 (donor+acceptor) transfected with the indicated siRNAs. The τ values are represented by a pseudocolor scale ranging from 1.8 to 2.5. Scale bar, 10 µm. (C and D) Quantification of average TGN-GFP lifetime in HeLa cells expressing TGN46-GFP and mCherry-Cb5 (donor+acceptor) and transfected with the indicated single (C) or combined (D) siRNAs. Donor indicates cells transfected with TGN46-GFP alone; CTRL indicates cells transfected with nontargeting siRNAs. The corresponding FRET efficiency is shown on the right. n = 20; data are means ± SD. *, P < 0.05; **, P < 0.01; Student’s t test. (E) Electron micrographs of ORP10-KD, OSBP1-KD, ORP9-KD, and OSBP1+ORP9-KD cells. (F) Corresponding quantification of the percentage of Golgi stacks engaged in ERTGoCS (ERTGoCS occurrence). n = 20–30 Golgi stacks. Scale bar, 200 nm.