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Sec24C is required for docking the prechylomicron transport vesicle with the Golgi

2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês

10.1194/jlr.m002758

ISSN

1539-7262

Autores

Shahzad Siddiqi, Shadab A. Siddiqi, Charles M. Mansbach,

Tópico(s)

Lipid Membrane Structure and Behavior

Resumo

The rate-limiting step in the transit of dietary fat across the intestinal absorptive cell is its exit from the endoplasmic reticulum (ER) in a specialized ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). PCTV bud off from the ER membranes and have unique features; they are the largest ER-derived vesicles (average diameter 250 nm), do not require GTP and COPII proteins for their formation, and utilize VAMP7 as a v-N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). However, PCTV require COPII proteins for their fusion with the Golgi, suggesting a role for them in Golgi target recognition. In support of this, PCTV contained each of the five COPII proteins when docked with the Golgi. When PCTV were fused with the Golgi, the COPII proteins were present in greatly diminished amounts, indicating they had cycled back to the cytosol. Immuno-depletion of Sec31 from the cytosol did not affect PCTV-Golgi docking, but depletion of Sec23 resulted in a 25% decrease. Immuno-depletion of Sec24C caused a nearly complete cessation of PCTV docking activity, but on the addition of recombinant Sec24C, docking activity was restored. We conclude that the COPII proteins are present at docking of PCTV with the Golgi and that Sec24C is required for this event. Sec23 plays a less important role. The rate-limiting step in the transit of dietary fat across the intestinal absorptive cell is its exit from the endoplasmic reticulum (ER) in a specialized ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). PCTV bud off from the ER membranes and have unique features; they are the largest ER-derived vesicles (average diameter 250 nm), do not require GTP and COPII proteins for their formation, and utilize VAMP7 as a v-N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). However, PCTV require COPII proteins for their fusion with the Golgi, suggesting a role for them in Golgi target recognition. In support of this, PCTV contained each of the five COPII proteins when docked with the Golgi. When PCTV were fused with the Golgi, the COPII proteins were present in greatly diminished amounts, indicating they had cycled back to the cytosol. Immuno-depletion of Sec31 from the cytosol did not affect PCTV-Golgi docking, but depletion of Sec23 resulted in a 25% decrease. Immuno-depletion of Sec24C caused a nearly complete cessation of PCTV docking activity, but on the addition of recombinant Sec24C, docking activity was restored. We conclude that the COPII proteins are present at docking of PCTV with the Golgi and that Sec24C is required for this event. Sec23 plays a less important role. The prechylomicron transport vesicle (PCTV) is an enlarged, COPII-containing vesicle that transports the developing chylomicron from the ER (endoplasmic reticulum) to the Golgi (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This largest of the lipoproteins is uniquely formed in the intestine and is the primary mechanism by which dietary fat is delivered to peripheral targeted tissues, muscle, heart, and adipose tissue. The majority of the triacylglycerol (TAG) in the chylomicron is derived from hydrolytic products of dietary lipids that are resynthesized to TAG by the intestinal ER. The newly synthesized TAG crosses the ER membrane and forms the prechylomicron in the ER lumen in a two-step process (3Cartwright I.J. Higgins J.A. Direct evidence for a two-step assemble of apoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes.J. Biol. Chem. 2001; 276: 48048-48057Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The exit step of the prechylomicron from the ER is the rate-limiting step by which dietary TAG traverses the intestinal absorptive cell (4Mansbach II, C.M. Nevin P. Intracellular movement of triacylglycerols in the intestine.J. Lipid Res. 1998; 39: 963-968Abstract Full Text Full Text PDF PubMed Google Scholar) (5Mansbach C.M. Dowell R. Effect of increasing lipid loads on the ability of the endoplasmic reticulum to transport lipid to the Golgi.J. Lipid Res. 2000; 41: 605-612Abstract Full Text Full Text PDF PubMed Google Scholar). Once detached from the ER membrane, the PCTV dock and then fuse with the Golgi using the N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex composed of VAMP7 (the R-SNARE), syntaxin5 (the Qa-SNARE), vti1A (the Qb-SNARE), and Bet1 (the Qc-SNARE) (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Our interest in the molecular mechanism of the docking of PCTV with the Golgi in relation to the COPII proteins was raised by these findings: (a) Although PCTV could be generated from ER membranes in the absence of Sar1, the initiator of the COPII complex, the vesicles formed could not fuse with the Golgi (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar). The vesicles were of the same size as PCTV generated using native cytosol, contained apolipoproteinB48 (apoB48), the quintessential chylomicron apolipoprotein, and VAMP7, the R-SNARE of the PCTV-Golgi SNARE complex; however, neither docking nor fusion was possible using these vesicles. b) The liver-fatty acid binding protein (L-FABP) can, in the absence of additional cytosolic components, elaborate PCTV from intestinal ER membranes (6Neeli I. Siddiqi S.A. Siddiqi S. Mahan J. Lagakos W.S. Binas B. Gheyi T. Storch J. Mansbach II, C.M. Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum.J. Biol. Chem. 2007; 282: 17974-17984Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, in support of the data obtained using vesicles formed in the absence of Sar1, these L-FABP formed vesicles do not contain COPII proteins and cannot fuse with the Golgi. In sum, the data strongly suggested to us that COPII proteins played a role in the docking process with the Golgi. Vesicles that take newly synthesized proteins from the ER to the Golgi are well described and require COPII proteins for their generation from ER membranes. These COPII proteins have been divided into an inner coat containing Sar1 and Sec23/24, and an outer coat containing Sec13/31 (7Fromme J.C. Orci L. Schekman R. Coordination of COPII vesicle trafficking by Sec23.Trends Cell Biol. 2008; 18: 330-336Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this construct, Sar1, in its GTP bound form, is recruited to the ER membrane and subsequently recruits Sec23/24 and then Sec13/31. These events occur in the presence of potential cargo for the vesicle at specific ER exit sites (ERES) (8Forster R. Weiss M. Zimmermann T. Reynaud E.G. Verissimo F. Stephens D.J. Pepperkok R. Secretory cargo regulates the turnover of COPII subunits at single ER exit sites.Curr. Biol. 2006; 16: 173-179Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 9Heinzer S. Worz S. Kalla C. Rohr K. Weiss M. A model for the self-organization of exit sites in the endoplasmic reticulum.J. Cell Sci. 2008; 121: 55-64Crossref PubMed Scopus (34) Google Scholar, 10Aridor M. Bannykh S.I. Rowe T. Balch W.E. Cargo can modulate COPII vesicle formation from the endoplasmic reticulum.J. Biol. Chem. 1999; 274: 4389-4399Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The completed structure then deforms the ER membrane and, in a fission step, is detached from the membrane. Most of these vesicles are remarkably uniform in size, ∼60 nm (11Bi X. Corpina R.A. Goldberg J. Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat.Nature. 2002; 419: 271-277Crossref PubMed Scopus (356) Google Scholar). Their size is constrained by the heterodimer Sec13/31, which forms a cage around the vesicle. However, new data from the Balch laboratory (12Stagg S.M. LaPointe P. Razvi A. Gurkan C. Potter C.S. Carragher B. Balch W.E. Structural basis for cargo regulation of COPII coat assembly.Cell. 2008; 134: 474-484Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) suggest that the angles of the cage components can be altered to some degree to allow for enlarging the cage to encompass larger cargos, up to 100 nm (12Stagg S.M. LaPointe P. Razvi A. Gurkan C. Potter C.S. Carragher B. Balch W.E. Structural basis for cargo regulation of COPII coat assembly.Cell. 2008; 134: 474-484Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). This is still not large enough to enable a chylomicron of 250 nm to be enclosed within the cage structure. By contrast, PCTV are large enough to contain chylomicrons and have been measured up to 350 nm (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar). The total data suggest that the COPII proteins are used by PCTV not as a vesicle generation mechanism but rather as a required Golgi targeting mechanism in addition to the R-SNARE VAMP7. As part of the targeting mechanism, the COPII proteins may not uncoat prior to the docking event of the PCTV with the Golgi. This report tests this hypothesis that has received support from recent data using COPII-dependent, protein vesicles (13Cai H. Yu S. Menon S. Cai Y. Lazarova D. Fu C. Reinisch K. Hay J.C. Ferro-Novick S. TRAPPI tethers COPII vesicles by binding the coat subunit Sec23.Nature. 2007; 445: 941-944Crossref PubMed Scopus (201) Google Scholar). 3H-Oleic acid (9.2 Ci/mM) was procured from Perkin Elmer Life Sciences. Immunoblot reagents were purchased from Bio-Rad. Enhanced chemiluminescence (ECL) reagents were procured from GE Healthcare. Protease inhibitor cocktail tablets were obtained from Roche Applied Science. Other biochemicals used were analytical grade and purchased from Sigma (Sigma Chemical Co., St. Louis, MO) or local companies. Male Sprague Dawley rats, 150–200 g were purchased from Harlan (Indianapolis, IN) and fed rat chow ad lib including the night prior to sacrifice. All research was done in accordance with NIH policies and was approved by the University of Tennessee Health Science Center Institutional Animal Use and Care Committee. Rabbit anti-Sar1 antibodies were raised commercially (Protein Tech Group, Chicago, IL) using recombinant Sar1 protein (Siddiqi et al., 2003). Polyclonal antibodies against rat VAMP7 were raised in rabbits against amino acids 105-123 of rat VAMP7 (14Siddiqi S.A. Mahan J. Siddiqi S. Gorelick F.S. Mansbach II, C.M. Vesicle-associated membrane protein 7 is expressed in intestinal ER.J. Cell Sci. 2006; 119: 943-950Crossref PubMed Scopus (49) Google Scholar). Affinity-purified rabbit polyclonal anti-Sec24C antibodies were a gift of J.P. Paccaud (University of Geneva, Geneva, Switzerland). Rabbit polyclonal antibodies against Sec13 and Sec31 were a generous gift of Dr. F. S. Gorelick (Yale University, New Haven, CT). Goat anti-Sec 23 antibodies and rabbit polyclonal antibody to syntaxin 5 were procured from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibodies to rBet1 were procured from Stressgen (Vancouver, Canada). Mouse anti-vti1a monoclonal antibodies were purchased from BD Biosciences. Rabbit polyclonal anti-apolipoprotein AI (apoAI) antibodies were a gift of Dr. Patrick Tso (University of Cincinnati, Cincinnati, OH). Sec24C recombinant protein was purchased from Novus Biologicals, Inc. (Littleton, CO). Goat anti-rabbit IgG conjugated with agarose beads was purchased from Sigma. Goat anti-rabbit IgG and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) were procured from Sigma. Enterocytes from the proximal half of male Sprague Dawley rat small intestine were isolated and radiolabeled with 3H-oleate as described (15Kumar N.S. Mansbach C.M. Determinants of triacylglycerol transport from the endoplasmic reticulum to the Golgi in intestine.Am. J. Physiol. 1997; 273: G18-G30PubMed Google Scholar). In brief, enterocytes were isolated from intestinal villi, collected, incubated with albumin bound 3H-oleate for 30 min at 35°C and washed twice with PBS containing 2% BSA to remove the excess 3H-oleate. The labeled enterocytes were homogenized using a Parr bomb and the ER was isolated using a sucrose step gradient that was repeated to purify the ER. The purified ER contained calnexin and calreticulin, markers proteins for ER, but neither GOS28 nor rab11, markers for Golgi and endosomes, respectively (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 14Siddiqi S.A. Mahan J. Siddiqi S. Gorelick F.S. Mansbach II, C.M. Vesicle-associated membrane protein 7 is expressed in intestinal ER.J. Cell Sci. 2006; 119: 943-950Crossref PubMed Scopus (49) Google Scholar). The Golgi was isolated from non-radiolabeled enterocytes as was cytosol (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 16Kumar N.S. Mansbach II, C.M. Prechylomicron transport vesicle: isolation and partial characterization.Am. J. Physiol. 1999; 276: G378-G386Crossref PubMed Google Scholar). Marker proteins assessed the purity of Golgi. Depletion of specific COPII proteins from intestinal cytosol was accomplished by incubating cytosol (1 mg protein) with 10μl of specific primary antibody at 4°C for 4 h. The antibody bound COPII proteins and any excess antibody were removed by incubation with anti-goat IgG bound to agarose beads or with anti-rabbit IgG bound to agarose beads at 4°C overnight and the beads removed by centrifugation. Depletion required two or three rounds of antibody treatment. Depletion was confirmed by immunoblot using specific antibodies. Depletion of specific ER proteins was accomplished by washing the ER membranes with 2M urea that was confirmed by immunoblot. In all studies using a specific COPII protein(s)-depleted system, both cytosol and ER were depleted. PCTV containing 3H -TAG was formed from 3H-TAG loaded intestinal ER (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar)). In brief, ER containing 3H -TAG (500 μg protein) was incubated at 37°C for 30 min with cytosol (1 mg protein) and an ATP-regenerating system in the absence of Golgi acceptor (total volume 500 µl). The incubation mixture was resolved on a continuous sucrose density gradient (0.1–1.15 M sucrose) and PCTV isolated from the light portions of the gradient. PCTV thus formed were concentrated using a Centricon-10 filter (Millipore Corp., Bedford, MA). Specific COPII protein depleted PCTV were generated by using specific COPII protein depleted cytosol and ER. For example, Sec23- or Sec24C-depleted PCTV were generated from Sec23- or Sec24C-depleted cytosol and ER. PCTV (150 μg protein) containing 3H-TAG were incubated with Golgi membranes (300 μg protein) and 500 μg cytosolic protein with 30 mM HEPES buffer containing 0.25M sucrose, 30 mM KCl, 5 mM CaCl2, 1 mM EDTA and 2 mM DTT for 1 h at 4°C (total volume 500 µl). The incubation mixture was resolved on a sucrose step gradient, and the Golgi fraction obtained by aspiration (14Siddiqi S.A. Mahan J. Siddiqi S. Gorelick F.S. Mansbach II, C.M. Vesicle-associated membrane protein 7 is expressed in intestinal ER.J. Cell Sci. 2006; 119: 943-950Crossref PubMed Scopus (49) Google Scholar). 3H-TAG was extracted (17Folch J. Lees M. Sloan-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) from the Golgi, and the radioactivity determined in a liquid scintillation spectrometer. To determine the effect of Sec23, Sec24C, or Sec31 depletion on PCTV-Golgi docking, Sec23-, Sec24C-, or Sec31-depleted cytosol and Sec23-, Sec24C-, or Sec31-depleted PCTV were used in the docking assay. These data were compared with PCTV docking activity after immuno-depletion with IgG. When recombinant Sec24C (rSec24C) was added back to Sec24C immuno-depleted cytosol for purposes of restoring docking activity, the rSec24C (7.25, 14.5, or 29 µg protein as indicated) was incubated with Sec24C immuno-depleted cytosol and Sec24C-depleted PCTV for 30 min at 4°C prior to starting the docking assay. The repleted cytosol and PCTV were used in the docking assay with Golgi membranes as described above. When anti-Sec24C antibodies were used in PCTV-Golgi docking experiments, native cytosol was used to generate PCTV. The native PCTV were incubated with anti-Sec24C antibodies (20 µl) for 40 min at 4°C prior to docking. The antibody-treated PCTV were then used in the docking assay with native cytosol and Golgi membranes. PCTV fusion with the Golgi was performed by incubating 3H-TAG loaded-PCTV (150 µg protein) with Golgi membranes (300 µg protein) and native cytosol (500 µg protein) for 30 min at 35°C with an ATP regenerating system, 5 mM MgCl2, 0.25M sucrose, 30 mM HEPES, pH 7.2, 30 mM KCl, 5 mM CaCl2, and 2 mM DTT; total volume, 500μl (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Postincubation, the Golgi membranes were separated from un-reacted PCTV by a sucrose step gradient, and the Golgi-associated TAG radioactivity was determined (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Isolation of the SNARE complex was accomplished by solubilizing PCTV docked with Golgi (200 μg) using 2% Triton X-100 in PBS as described (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In brief, the solubilized proteins were incubated overnight at 4°C with anti-rabbit VAMP7 antibody bound to anti-rabbit IgG conjugated with agarose beads. The beads were collected by centrifugation, washed eight times with PBS to remove unbound proteins/antibodies, and resuspended in Laemmle's buffer for immunoblot analysis. The beads were either boiled or not boiled in Laemmle's buffer as indicated. The immuno-precipitated proteins were separated by SDS-PAGE, then transblotted onto nitrocellulose membranes (Bio-Rad). The same membrane was probed with antibodies to VAMP7, syntaxin 5, vti1a, and rBet1. In experiments where the ability of rSec24C to reestablish Sec24C depleted PCTV-Golgi docking was tested, 29 µg rSec24C were incubated with Sec24C depleted-PCTV (150 µg prot) and Sec24C depleted-cytosol (500 µg prot) for 60 min at 4°C. The Sec24C repleted PCTV were then incubated with Golgi membranes (300 µg prot) as for docking and the SNARE complex isolated as described (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Proteins were separated by SDS-PAGE, then transblotted onto nitrocellulose membranes (Bio-Rad) (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar). After incubation with specific primary antibodies and peroxidase-conjugated secondary antibodies, proteins were detected by ECL (Amersham Biosciences) and Biomax film (Eastman Kodak, Rochester, NY). TAG radioactivity was determined by liquid scintillation spectroscopy (15Kumar N.S. Mansbach C.M. Determinants of triacylglycerol transport from the endoplasmic reticulum to the Golgi in intestine.Am. J. Physiol. 1997; 273: G18-G30PubMed Google Scholar). In contrast to older information (18Barlowe C. d'Enfert C. Schekman R. Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum.J. Biol. Chem. 1993; 268: 873-879Abstract Full Text PDF PubMed Google Scholar), more recent data would suggest that ER-to-Golgi transport vesicles which contain COPII proteins are attached to Golgi tethering proteins prior to their eventual fusion with the Golgi (13Cai H. Yu S. Menon S. Cai Y. Lazarova D. Fu C. Reinisch K. Hay J.C. Ferro-Novick S. TRAPPI tethers COPII vesicles by binding the coat subunit Sec23.Nature. 2007; 445: 941-944Crossref PubMed Scopus (201) Google Scholar). We wished to determine if that were true for PCTV and Golgi membranes in the intestine. To this end, we incubated PCTV, which contain all the COPII proteins (Fig. 1A, lane 1) with Golgi under conditions that favor docking (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Although the Golgi gave a weak signal for each of the COPII proteins prior to incubation (Fig. 1A, lane 2), after a 60 min incubation with PCTV, the COPII proteins in the Golgi gave a much stronger signal (Fig. 1A, lane 3). One interpretation of these data is that the COPII proteins on PCTV do not un-coat prior to docking with the Golgi and thus remain during the docking process resulting in their increased concentration on isolation of the Golgi. Under conditions in which fusion with the Golgi would be expected (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), the signals for all the COPII proteins were greatly diminished (Fig. 1A, lane 4), suggesting that the COPII proteins returned to cytosol after fusion. To establish that PCTV were fused with the Golgi, we took advantage of our prior observation that prechylomicrons in PCTV do not contain apoAI (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar). However, apoAI is present in the Golgi lumen (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and becomes associated with the prechylomicrons when they enter the Golgi lumen after PCTV-Golgi fusion (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In the current experiments, as before, prechylomicrons in PCTV contain no apoAI (Fig. 1B, chylo), and the few chylomicrons isolated from the Golgi lumen by carbonate treatment show small amounts of apoAI (Fig. 1B, Golgi). By contrast, under conditions where fusion of the PCTV with the Golgi is expected, the Golgi lumen becomes enriched with chylomicrons and the amount of apoAI associated with them is greatly (3-fold) increased (Fig. 1B, Golgi post fusion), supporting our prior data (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Our next aim was to determine which of the COPII proteins was primarily important for target recognition for PCTV with the Golgi. We have previously shown that in the absence of Sar1, PCTV do not fuse with the Golgi (1Siddiqi S.A. Gorelick F.S. Mahan J.T. Mansbach II, C.M. COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle.J. Cell Sci. 2003; 116: 415-427Crossref PubMed Scopus (119) Google Scholar, 6Neeli I. Siddiqi S.A. Siddiqi S. Mahan J. Lagakos W.S. Binas B. Gheyi T. Storch J. Mansbach II, C.M. Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum.J. Biol. Chem. 2007; 282: 17974-17984Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We next considered Sec23. As expected, intestinal cytosol contained significant amounts of Sec23 as shown by its strong signal on immunoblot (Fig. 2A, lane 1). By contrast, using bead-bound specific anti-Sec23 antibodies, Sec 23 was completely removed from the cytosol by the immuno-depletion (Fig. 2A, lane 2). PCTV generated in IgG-depleted cytosol contained Sec23 (Fig. 2A, lane 3), whereas PCTV formed in Sec23-depleted cytosol did not (Fig. 2A, lane 4), as expected. Sec24C was modestly reduced by the immuno-depletion of Sec23 (Fig. 2A, Sec24C). When 3H-TAG loaded PCTV generated in IgG immuno-depleted cytosol were incubated with Golgi membranes under conditions favoring docking (2Siddiqi S.A. Siddiqi S. Mahan J. Peggs K. Gorelick F.S. Mansbach II, C.M. The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.J. Biol. Chem. 2006; 281: 20974-20982Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), a robust 3H-TAG signal was obtained in the Golgi fraction, indicating significant amounts of docking (Fig. 2B, IgG). However, when PCTV generated in Sec23-depleted cytosol were incubated with Golgi under conditions favoring docking, a 25% reduction in the ability of the Sec23-depleted PCTV to dock with the Golgi was found (Fig. 2A, Sec23 dep). The reduced docking activity of the Sec23-depleted cytosol may be due to partial Sec24C reduction as a result of the Sec23 depletion (Fig. 2A, Sec24C). An interpretation of these data is that Sec23, while important as a Golgi targeting signal, is not required for docking to occur. Because immuno-depletion of Sec23 seemed to have only a modest effect on PCTV-Golgi docking, we immuno-depleted Sec24C from cytosol using specific antibodies. This resulted in the removal of Sec24C from the cytosol (Fig. 3A, compare IgG depleted cytosol with Sec24C depleted cytosol). Densitometric analysis revealed that some (73%) Sec23 remained (Fig. 3A, Sec24C depl), presumably because only Sec24C was removed, leaving Sec23 potentially complexed to the remaining Sec24 paralogs (Sec24A, B, or D). When the Sec24C-depleted cytosol was used in place of IgG-depleted cytosol in the generation of PCTV from Sec24C-depleted ER membranes, the PCTV produced were also devoid of Sec24C, as expected (Fig. 3A, compare PCTV formed in IgG- depleted cytosol with PCTV formed in Sec24C-depleted cytosol). Impo

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