Volume 11, Issue 5, Pages 671-682 (November 2006) Phosphatidylinositol 4-Phosphate Formation at ER Exit Sites Regulates ER Export  Anna Blumental-Perry, Charles J. Haney, Kelly M. Weixel, Simon C. Watkins, Ora A. Weisz, Meir Aridor  Developmental Cell  Volume 11, Issue 5, Pages 671-682 (November 2006) DOI: 10.1016/j.devcel.2006.09.001 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The Fapp1-PH Domain Inhibits ER Export (A) Schematic representation of COPII coat assembly and ER export. The release of cargo proteins to the vesicular fraction was measured by differential centrifugation. M designates medium-speed donor-membrane-containing pellet (16,000 × g). H designates high-speed vesicle containing pellet (186,000 × g). (B–D) VSV-Gts-containing microsome membranes were incubated with cytosol for 30 min at 32°C in the presence or absence of neomycin (1.5 mM) (B) or the indicated GST-tagged PH domains (C and D), and the release of VSV-Gts (B and C) or Bet1 (D) to the vesicle fraction was analyzed. (E–G) VSV-Gts-expressing microsomes were incubated with cytosol (E) or purified COPII components (Sar1 [1 μg], Sec23/24 [0.4 μg], and Sec13/31 [6 μg], 40 μl final volume) (F and G) in the presence of PLCδ1-PH (E and F) or Fapp1-PH (G) as indicated for 30 min at 32°C. The release of VSV-Gts to the vesicular fraction was analyzed. The results are representative of at least two independent experiments. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 PtdIns4P Binding Mediates Fapp1-PH Inhibition of ER Export (A) VSV-Gts-infected microsome membranes were incubated with purified COPII components (as in Figure 1) in the presence of GST-Fapp1-PH domain as indicated for 30 min at 32°C. The mobilization of VSV-Gts to the vesicular fraction and the presence of ARF1 in the analyzed fractions were determined by western blot. Recombinant ARF1 was loaded separately as a control. (B) VSV-Gts-containing microsomes were incubated with cytosol in the presence of the indicated concentrations of GST-Fapp1-PH domain with or without addition of liposomes (Lpsm) composed of PC or PC/PtdIns4P (420/50 μM) for 30 min at 32°C as indicated. The release of VSV-Gts to the vesicular fraction is quantified in the graph below. Data are averaged from three independent experiments (means ± SD). (C) VSV-Gts-expressing microsomes were incubated with cytosol in the presence or absence of GST-Fapp1-PHWT or GST-Fapp1-PHNK for 30 min at 32°C, and the budding of VSV-Gts was determined. (D) Quantitation of VSV-Gts released into vesicular fractions in the presence of varying concentrations of GST-Fapp1-PHWT and Fapp1-PHNK domains. Data are means ± SD derived from five independent experiments. (E) VSVts-infected cells were permeabilized and incubated in the presence or absence of rat liver cytosol (RLC) GST-Fapp1-PHWT (3 μM) or GST-Fapp1-PHNK (5 μM) as indicated for 30 min at 32°C. The mobilization of VSV-Gts from reticular ER (upper left image) to VTCs (upper right image) was determined by IF. The images are a projection of five consecutive 0.1 μm optical sections. Scale bar is 5 μm. (F) Liposomes (40 μl) composed of phosphatidylserine (500 μM) and PtdIns4P (100 μM) were incubated at 32°C for 3 min with indicated amounts of the cytosolic domain (amino acid 1–507) of Sac or SacC392S mutant. Reactions were terminated by the addition of NEM (50 mM). Phosphate release was quantified by a malachite green assay. Data are means ± SD of triplicate samples. (G) VSV-Gts microsomes (40 μl final volume) were incubated with cytosol in the presence or absence of of Sac1 (0.9 μg) or Sac1C392S (3.6 μg) as indicated for 30 min at 32°C and the mobilization of VSV-Gts to the vesicle fraction was determined. The results are representative of at least three independent experiments. (H) VSVts infected cells were permeabilized and incubated (220 μl final volume) in the presence or absence of RLC and Sac1 (1 μg) as indicated for 30 min at 32°C. The movement of VSV-Gts from reticular ER (upper image) to VTCs (middle image) and the effects of Sac1 on VSV-Gts mobilization (lower image) were determined by IF. Quantitation of VSV-Gts transport to VTCs at 20 and 30 min incubations in the presence or absence of the indicated amounts of Sac1 is shown on the right panel. The number of VSV-Gts containing VTCs was derived from analysis of random fields of cells by automatically counting 100 cells or more for each data point. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Sar1 Recruitment and Activation on ER Membranes Stimulates PtdInsP and PtdInsP2 Formation (A) ER enriched membrane fractions were incubated with buffer (control) or with Sar1-GTP (H79G) (2 μg, 60 μl final volume) for 15 min at 32°C. Membranes were washed and incubated in the presence of γ-32P-ATP and cytosol for the indicated times at 32°C. At the end of incubations, lipids were extracted and separated by TLC. Phospholipids were quantified on a phosphor imager, and lipid markers were identified by iodine vapors. (B) Quantitation of (A) (means ± SEM derived from four independent experiments). (C) ER enriched membrane fractions, were incubated with buffer (control), Sar1-GTP, or Sar1-GDP (T39N, 2 μg of each, 60 μl final volume) for 15 min at 32°C. Membranes were washed and further incubated for 3 min as described in (A). Data are means ± SD of three independent experiments. (D) Membranes were incubated and lipids extracted as described in (C) except that orthovanadate (200 μM) was included in the second incubation to inhibit phosphatase activities. (E) Quantitation of (D). Data represent means ± SD of four independent experiments. (F) Membranes were incubated in the absence or presence of Sar1-GTP (6 μg, 40 μl final volume) with γ-32P-ATP in the presence or absence of wortmannin (5 μM) or adenosine (500 μM) for 9 min at 32°C as indicated. Lipids were extracted and processed as described above. (G) The effect of indicated inhibitors (analyzed as in [F]) was calculated by dividing the fold stimulation of PtdInsP formation by Sar1 (over control) in the presence or absence of inhibitors, at time points at which peak PtdInsP formation was observed. Data are mean ± SD of three to five independent experiments. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Dynamics of PtdIns4P Formation at ER Export Sites Infected cells that accumulated VSV-Gts at 40°C in the ER were permeabilzed and incubated on ice for 20 min in the presence of an Alexa 594-labeled Fab fragment of a monoclonal antibody to VSV-Gts. The cells were washed and shifted to 32°C to initiate ER export in the presence of cytosol and GFP-Fapp1-PH-domain (0.2 μM). Fluorescence pair images were acquired every 2 min by time-lapse spinning disc confocal video microscopy. Sample times (min) are indicated for each panel on the right. Arrows indicate colocalization between VSV-Gts and PtdIns4P at ERES. The area labeled with the large arrowhead in (B), (C), and (D) is enlarged in the inserts. Note colocalization of VSV-Gts and PtdIns4P at sites of initial VSV-Gts concentration (B and C) during the mobilization of VSV-Gts from the reticular ER (A) to mature VTCs (E). Transient colocalization of PtdIns4P reporter with VSV-Gts is mainly observed in the initial stages of VSV-Gts trimerization and ER export (B and C). Scale bar is 5 μm. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 The Recruitment of Sec23 to ERES Is Assisted by PtdIns4P (A) Permeabilized cells were incubated in the presence of Sar1-GTP (6 μg, 220 μl final volume), RLC, and GFP-Fapp1-PH (3 μM) for 30 min at 32°C. The cells were fixed and stained for Sec23 and mannosidase II (Man II) as indicated. The localization of GFP-Fapp1-PH (by GFP fluorescence) ERES (Sec23, red) and the Golgi complex (Man II, blue) was determined. Merge images for Man II and GFP-Fapp1-PH, Sec23 and Man II, and a merged image for all three markers are shown. Arrows indicate colocalization of Fapp1-PH with peripheral Sec23-containing ERES. Scale bar is 5 μm. (B) Microsomes were incubated with purified Sec23/24 complex (0.7 μg, 60 μl final volume) and 1 mM ATP at 32°C for 15 min in the presence or absence of Sar1-GTP (0.25 μg) and GST-Fapp1-PH domain as indicated. The membrane recruitment of Sec23 was determined by western blot with affinity-purified anti-Sec23 antibody. (C) Microsomes were washed in buffer containing 2.5 M urea for 30 min on ice. The membranes were reisolated by centrifugation and incubated with RLC and 1 mM ATP as in (B), in the presence or absence of Sar1-GTP (0.1 μg) and GST-Fapp1-PH as indicated, and membrane recruitment of Sec23 was determined. (D) Microsomes were incubated with RLC at 32°C for 15 min in the presence or absence of Sar1-GTP (0.3 μg, 60 μl final volume), ATP-regeneration system (indicated as reg.), 1 mM ATP, or 50 μM PtdIns4P micelles as indicated, and the recruitment of Sec23 was determined. Data are representative of three independent experiments. (E–F) Permeabilized cells (220 μl final volume) were incubated with RLC and ATP-regeneration system in the presence or absence of Sar1-GTP (6 μg) and GST-Fapp1-PH domain (5 μM) (E) or neomycin (1 mM) (F) as indicated for 30 min at 32°C. The cells were fixed and stained for Sec23 (E) or Sec23 and Sec13 (F). Images for each experiment were acquired under identical conditions. Scale bar is 5 μm. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 PtsIns4P Is Formed and Accumulated on Sar1-Induced Tubules (A) Permeabilized cells (220 μl final volume) were incubated in the presence of Sar1-GTP (6 μg) for 30 min at 32°C. The cells were fixed and stained for Sar1 and giantin (merged images are shown as indicated). (B and C) VSVts-infected cells were permeabilized cells and incubated as in (A) in the presence of 0.6 μM (B) or 5 μM (C) of GST-Fapp1-PH for 30 min at 32°C to form VSV-Gts-containing tubular ERES. The cells were fixed and stained with VSV-Gts and GST-Fapp1-PH as indicated. (D) Permeabilized cells were incubated with anti-PtdIns4P antibodies as in (A). The cells were fixed and further stained for VSV-Gts. The localization of VSV-Gts and the anti-PtdIns4P antibody was determined by IF. A projection of five consecutive optical sections is presented. Bars are 10 μm. Note the colocalization of VSV-Gts and PtdIns4P reporters on Sar1 tubular domains (arrow in [B]). Arrowheads in (B) point to juxtanuclear Golgi elements positive for Fapp1-PH. (E) Permeabilized cells were incubated as in (A) in the presence or absence of neomycin (1 mM). The cells were fixed stained for Sar1. Note inhibition of Sar1 tubule elongation with neomycin or Fapp1-PH. Developmental Cell 2006 11, 671-682DOI: (10.1016/j.devcel.2006.09.001) Copyright © 2006 Elsevier Inc. Terms and Conditions