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First published online 4 December 2002
doi: 10.1242/jcs.00215

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COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle

¹ Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
² Veterans Affairs Medical Center, Memphis, TN 38104, USA
³ VA Connecticut Healthcare, West Haven, CT 06516, USA
⁴ Department of Medicine, Yale University School of Medicine, New Haven, CT 06520, USA

View larger version (24K): [in a new window]   Fig. 2. Effect of cytosol and ATP on PCTV and protein vesicle budding from ER. (A) ER containing 3H-TAG was incubated in the presence (+) or absence (-) of intestinal cytosol. A continuous sucrose gradient (0.1-1.15 M) was used to isolate the PCTVs. The total 3H-dpm from each fraction (150 µl of 500 µl total volume) was counted. (B) A similar experiment to A was performed with (+) or without (-) ATP and an ATP generating system. The gradient was resolved as in A and total 3H-dpm determined. In A and B only fractions 1-20 of 70 total fractions (total tube volume, 35 ml) are shown since no 3H-dpm above baseline were found in fractions 21-70. (C) ER containing 14C-TAG and 3H-protein were incubated with and without cytosol in the presence of an ATP-generating system. The reaction mix was resolved on the sucrose gradient using 11 ml centrifuge tubes and fractions of 0.5 ml collected.

View larger version (103K): [in a new window]   Fig. 1. (A) Negative stained EM of PCTVs generated from intestinal ER. PCTVs (fractions 1-7 from Fig. 2A) were formed from intestinal ER and examined by EM. All the vesicles appear intact and range in size from 142-347 nm (12,000x). The bar represents 500 nm. (B) Thin section transmission microscopy of PCTVs and protein vesicles (inset). PCTVs and protein vesicles were stained and processed for thin section EM (5000x). PCTVs are 350-500 nm in size and contain osmiophilic lipid. Bar, 500 nm. (Inset) Protein vesicles (fractions 9-12 from Fig. 2C, prepared for EM appeared as 70-117 nm in diameter and lacked lipid. Bar, 100 nm. For details, see Materials and Methods.

View larger version (26K): [in a new window]   Fig. 5. (A) PCTVs and protein vesicles concentrate SNARE proteins as analyzed by immunoblotting of different subcellular fractions for syntaxin 5 and p58. Proteins (30 µg from each fraction) were separated on 12% SDS-PAGE and immunoblotted as described. (B) The effect of protease treatment on a PCTV cargo protein. PCTVs were incubated with proteinase K (0.5 mg/ml) in the presence or absence of 1% Triton X-100 for 30 minutes at 4°C. 30 µg of protein from treated or untreated PCTVs were used for immunoblotting for apoB-48. (C) Immunoblots of subcellular fractions for Sar1, Sec13 and Sec31. Proteins (30 µg for each fraction except PCTVs, 10 µg) from the different subcellular fractions were separated on 12% SDS-PAGE (Sar1 and Sec13) and 10% SDS-PAGE (Sec31), followed by immunoblotting as in Fig. 2. (D) Immunoblot showing depletion of Rab1 from cytosol (Rab1 dep cy) by using anti-Rab1 antibodies and depletion of Rab1 from the ER (Rab-GDI ER) by treatment with Rab-GDI. For details, see Materials and Methods.

View larger version (35K): [in a new window]   Fig. 3. Immunoblots of subcellular fractions using antibodies to apoB-48, apoA-I, apoA-IV (A), and calnexin and calreticulin (B). 30 µg of protein from each fraction were separated on 7.5% (for apoB) or 10% (for other indicated proteins) SDS-PAGE.

View larger version (24K): [in a new window]   Fig. 4. Transport to the Golgi and delivery to the Golgi lumen of newly synthesized 14C-TAG and 3H-apoB-48 in PCTVs. (A) PCTVs (150 µg prot) containing 14C-TAG and 3H-apoB-48 were incubated with non-radiolabeled Golgi (300 µg prot) in the presence (+) or absence (-) of cytosol (0.5 mg prot). The Golgi was separated from PCTVs and the Golgi 14C-TAG extracted. The Golgi 3H-apoB-48 was isolated by immunoprecipitation. Of the total 14C-TAG-dpm and 3H-apoB-48-dpm, 28% and 25%, respectively, were delivered to the Golgi during the incubation. (B) After the fusion reaction, the Golgi was separated from PCTV on a sucrose gradient and incubated with or without carbonate (100 mM, pH 11) as indicated to release the chylomicrons. Chylomicron 14C-TAG was extracted, and its 3H-apoB-48 isolated by immunoprecipitation. The data are the means of three experiments. (C) 3H-dpm-PCTVs (150 µg prot, 30,640 dpm) were incubated with Golgi membranes (300 µg prot). The Golgi were isolated and treated with (+) or without (-) trypsin (0.5 mg/ml final concentration) for 1 hour at 4°C. Trypsin inhibitors were added and the Golgi isolated. 3H-dpm was determined for each fraction. (Inset) 30 µg protein from fractions 18-21 from the incubations with or without trypsin were loaded onto SDS-PAGE and immunoblotted for GOS28. (D) The fusion of 3H-TAG-PCTV generated in native cytosol (Normal PCTVs) or Sar1-depleted cytosol (COPII dep PCTV) with Golgi. The Golgi was isolated and 3H-TAG extracted. The data are the mean±1 s.e.m. (n=3). For details, see Materials and Methods.

View larger version (9K): [in a new window]   Fig. 6. Immunoblot showing the distribution of Sar1, Sec31, rBet1, apoB-48 and calnexin across a sucrose gradient. ER, cytosol and an ATP regenerating system were incubated for 30 minutes at 37°C and placed on a sucrose gradient as in Fig. 2C. The gradient was resolved and an equal volume (100 µl) of each fraction and 20 µl (60 µg protein) of the original ER and cytosol mixture (TM) were separated on SDS-PAGE. The gel was transblotted and probed with antibodies to the indicated proteins.

View larger version (13K): [in a new window]   Fig. 9. Effect of Sar1-depleted cytosol on the presence of COPII proteins in PCTVs. (A) Native cytosol (Cy), Sar1 immunodepleted cytosol (Sar1 dep Cy), native ER, and ER washed with 2 M urea (Urea-treated ER) (30 µg protein each) were probed with anti-Sar1 antibodies. (B) PCTVs (30 µg protein), formed under different conditions as indicated, were used for immunoblots as in Fig. 2. PCTVs formed in native cytosol (Cy) or from Sar1 immunodepleted cytosol (Sar1 dep Cy) or in the presence of H89 (H89) were probed with anti-Sar1 antibodies. PCTVs (30 µg protein) formed in native cytosol (Cy) or Sar1 immunodepleted (Sar1 dep Cy) cytosol were probed with antibodies to Sar1, Sec24, Sec31, syntaxin 5, p58 and rBet1. (C) The effect of protease treatment on PCTV cargo (apoB-48) generated in Sar1-depleted cytosol. PCTVs generated in Sar1 depleted cytosol were incubated with proteinase K (0.5 mg/ml) as in 5B. 30 µg protein of protease treated or untreated PCTVs were separated on 7.5% SDS-PAGE and immunoblotted with rabbit polyclonal anti-apoB-48 antibodies. For details, see Materials and Methods.

View larger version (21K): [in a new window]   Fig. 7. The effect of Sec31 antibodies on PCTV budding. The budding assay was performed in the absence (-) or presence (+) of native cytosol (1 mg protein) plus pre-immune IgG and cytosol (1 mg protein) treated with 3 µl of anti-Sec31 antibodies (Sec31). Data from fractions 1-20 are shown since fractions 21 to 70 had no 3H-dpm over baseline.

View larger version (42K): [in a new window]   Fig. 8. The effect of Sar1 antibodies and Sar1-depleted cytosol on PCTV (A,B,C, E) and protein vesicle (D,F) budding from ER. (A) The effect of Sar1-depleted cytosol (0.8 mg protein) on PCTV-budding. Native ER was incubated with (+) or without (-) cytosol and compared with ER incubated with Sar1-depleted cytosol (0.8 mg protein). Total 3H-dpm was determined for each fraction. (B) Cytosol (1 mg protein) was (+) or was not (-) included in the budding assay. Pre-immune IgG was added to the native cytosol (+ Cytosol + IgG). Native ER was incubated with cytosol to which Sar1 antibodies had been added (ER + Sar1 Ab.) Native ER was treated with 2 M urea and incubated with cytosol to which Sar1 antibody had been added (Urea ER + Sar1 Ab). The total 3H-dpm is shown on the ordinate. For A and B, 35 ml centrifuge tubes were used. Data from fractions 1-20 are shown since fractions 21 to 70 had no 3H-dpm over baseline. (C,D) The effect of Sar1-depleted cytosol on PCTV and protein vesicle budding. ER containing 14C-TAG and 3H-protein was immunodepleted of Sar1 (+ Sar1 dep cy), mock depleted cytosol (Moc dep cy) or no cytosol (- Cy). The reaction mix was resolved on a sucrose gradient using 11 ml centrifuge tubes and 0.5 ml fractions obtained. The 14C-dpm-TAG (C) and TCA precipitable 3H-dpm-protein (D) were determined for each fraction. (E,F) The effect of recombinant Sar1 protein on PCTV and protein vesicle budding in the presence of Sar1-depleted cytosol. Experiments similar to C and D were performed using 14C-TAG and 3H-protein-labeled ER incubated with no cytosol (- Cy), mock depleted cytosol (+ Cy), Sar1-depleted cytosol (Sar1 dep cy) or Sar1-depleted cytosol supplemented with recombinant Sar1 protein (Sar1 rep cy). 14C-TAG-dpm (E) and 3H-protein-dpm (F) were determined.

View larger version (83K): [in a new window]   Fig. 10 Negatively stained and thin section electron microscopy of PCTVs formed in the presence of Sar1-depleted cytosol and immunoelectron microscopy of these PCTVs using syntaxin 5 and rBet1 antibodies. PCTVs were formed in Sar1-immunodepleted cytosol and isolated. (A) The PCTVs were processed as in Fig. 1A. Intact vesicles ranging from 150 to 350 nm are seen. Bar, 500 nm. (B) The PCTVs were examined by thin section electron microscopy as in Fig. 1B. The PCTV were 300-350 nm in diameter. Bar, 200 nm. (C) Negatively stained PCTVs as in Fig. 1A were incubated with mouse anti-rBet1 (10 nm gold particles) and rabbit anti-syntaxin-5 (15 nm gold particles) antibodies. Representative views are shown in i-iii. In iv, pre-immune IgG was used. For details, see Materials and Methods.

View larger version (24K): [in a new window]   Fig. 11. The effect of GTPS and H89 on PCTV and protein vesicle budding. 500 µg ER protein was incubated with (+) or without (-) cytosol or cytosol to which GTPS (200 µM) or H89 (200 µM) was added as indicated in the key. (A) The total 14C-TAG-dpm of each 0.5 ml fraction was determined. (B) The total 3H-TCA precipitable protein-dpm was measured in each 0.5 ml fraction.

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