Quantification was performed as in Figure 1D. (d-Cer-C6) for 2.5 h and further incubated with fresh medium containing each reagent for 3 h. Western blotting and quantification were performed as in D. Asterisk indicates 0.0005. VAPs regulate PAUF processing and secretion via lipid transfer between the ER and the Golgi complex Ceramide transported by VAP/CERT is converted, together with PC, to SM and DAG by SM synthase at the 0.02 (B) and 0.005 (C). (D and E) HEK 293T cells were transfected with a PAUF-MycHis plasmid alone (Cont) or with a PAUF-MycHis plasmid in combination with a plasmid for the wild type (WT) or the FF/AA mutant of HA-CERT (D) or Myc-OSBP (E). After 20 h, the cells were washed and incubated with fresh medium for 4 h. The medium and the cell lysate were Western blotted with the indicated antibodies. Quantification was performed as in Figure 1D. Asterisk in D denotes a nonspecific polypeptide, and asterisk in E indicates 0.02. VAPs are required for membrane fission during CARTS biogenesis Our data so far obtained raise the possibility that VAPs regulate PAUF processing and secretion through at least two different events: 1) DAG production followed by PKD recruitment to the TGN, and 2) cholesterol- and SM-rich microdomain organization at the TGN. To investigate the roles of VAP-mediated lipid signaling in the biogenesis of CARTS, we visualized PAUF-MycHisCcontaining CARTS in HeLa cells by fluorescence microscopy. As shown previously (Wakana = 102C123 cells per condition) was determined, and the average values of four independent experiments Pictilisib dimethanesulfonate Pictilisib dimethanesulfonate are shown (mean SD). Asterisks indicate 0.0001. (C) Quantification of PAUF-MycHisCcontaining CARTS. The number of CARTS in 25-OHCtreated control cells (= 1208 punctate elements in 10 cells) and VAP-A/B-depleted cells with or without large tubules (tub) (= 432 and 850 punctate elements in 10 cells, respectively) was determined (mean SD). Asterisk indicates 0.0001. (D and E) HeLa cells were transfected with control siRNA or a mixture of siRNA oligos specific for VAP-A (523) and VAP-B (498). At 72 h after siRNA transfection, the cells were treated with 2 g/ml 25-OH for 2.5 h and subjected to CARTS formation assay as described in Rabbit polyclonal to ZFP161 0.03. VAPs are required for the biogenesis of CARTS in permeabilized cells We further investigated the effects of VAP-A/B knockdown on CARTS biogenesis by using a biochemical assay that we established previously (Wakana polarity (Cole = 202C250 cells per condition) in the presence or absence of 25-OH was determined, and the average values of three independent experiments are shown (mean SD). Asterisks indicate 0.0001. Altogether these results suggest the presence of VAP-OSBP-Sac1 and Pictilisib dimethanesulfonate VAP-CERT complexes at the specialized ER subdomains closely apposed to the 0.02. (B and C) HeLa cells were cotransfected with a plasmid for FAPP-PH-GST (B) or Myc-OSBP (C) in combination with a plasmid for GFP-Sac1 wild type or C/S, fixed, and visualized with GFP and an antibody against GST or Myc. Pictilisib dimethanesulfonate Scale bars: 10 m. AssociationCdissociation dynamics of ERCGolgi contacts are important for CARTS biogenesis Antonny and colleagues (Mesmin 0.04. (C) HeLa cells stably expressing PAUF-MycHis were transfected with a plasmid for mRFP (control) or PH-FFAT-mRFP, fixed, and visualized with mRFP and an anti-Myc antibody. Scale bar: 10 m. (D) Quantification of PAUF-MycHisCcontaining CARTS. The number of CARTS in mRFP-expressing cells (= 1128 punctate elements in 10 cells) and PH-FFAT-mRFP-expressing cells (= 289 punctate elements in 10 cells) was determined (mean SD). Asterisk indicates 0.0001. DISCUSSION Our work revealed that VAP-A/B knockdown impaired the processing and secretion of PAUF, which is one of the cargo proteins of CARTS. Similar effects were caused by d-ceramide-C6 treatment or disruption of the functions of CERT and OSBP, suggesting the requirement of VAP-mediated nonvesicular lipid transfer between the ER and the Golgi complex for PAUF processing and secretion. An in vivo analysis and an analysis Pictilisib dimethanesulfonate with biochemical CARTS formation assay showed that VAPs are required for CARTS biogenesis at the TGN. A series of our experiments suggest that VAPs form complexes with CERT and OSBP/Sac1, respectively, at specialized ER subdomains where the (low speed) and at 100,000 (high speed). The pellet was Western blotted with an anti-TGN46 antibody to estimate the amount of CARTS generated. Live-cell imaging HeLa cells stably expressing GFP-Sac1 were transfected with a plasmid for mRFP or PH-FFAT-mRFP. After 20 h, the medium was replaced with Opti-MEM, and cells were maintained in 5% CO2 at 37C during live-cell.