EXPERIMENTAL

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RESEARCH

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Biogenesis of Endoplasmic Reticulum Transport Vesicles Transferring Gastric Apomucin from ER to Golgi AMALIASLOMIANY,~ EWAGRZELINSKA,CHINNASWAMYKASINATHAN,KEN-ICHIROYAMAKI, DANUTAPALECZ,BEATRIX A. SLOMIANY,ANDBRONISLAW L. SLOMIANY Research

Center,

of Medicine

University

and

Dentistry

of New

Jersey,

Uniuersity

reprint

requests

should

Street,

Newark,

New

Jersey

07103-2400

PROCEDURES

Materials. [‘4C]Phosphocholine, cytidine 5’.diphospho-[m&ylyl3H]choline, and [y-32P]ATP were purchased from Amersham Corp. (Arlington Heights, IL). The standard phospholipids were from Avanti Polar Lipids (Birmingham, AL). The CAMP-dependent protein kinase (catalytic subunit), alkaline phosphatase bound to agarose, and specific biochemical compounds were from Sigma Chemical Co. (St. Louis, MO). Antibodies against the apomucin precursor and protein fatty acyltransferase were prepared in our laboratory [12,13]. Anti-ribophorin monoclonal antibody (olR1 MAb) was kindly provided by Dr. Kreibich (Rockefeller IJniversity, NY) and the authentic NSF was a gift from Dr. C. Beckers (Sloan-Kettering Institute, NY). Other reagents and chemicals were obtained from Bio-Rad (Rockville Centre, NY), Pharmacia LKB Biotechnology (Piscataway, NJ), Aldrich Chemical (Milwaukee, WI), Fisher Scientific (Springfield, NJ), and GIRCO (Grand Island, NY). Preparation of endoplasmic reticulum and Golgi membranes. Rat gastric mucosal tissue (50 g) was placed in 5 vol of ice-cold phosphate-

The movement of newly synthesized proteins from the endoplasmic reticulum (ER) to Golgi stock and through successive processing compartments of the Golgi apparatus is accomplished by repeated rounds of budding and fusion of transport vesicles [l-3]. Significant progress toward understanding of vesicular transport through the secretory pathway of eukaryotic cells was achieved by the development of an in vitro assay on and

Bergen

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INTRODUCTION

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semi-intact cells [4, 51 and cell-free systems that reconstitute vesicular transfers [6-91. The results of these investigations provided evidence that a single round of vesicular transport between ER and Golgi involves Nethylmaleimide-sensitive, GTPyS-sensitive, ATP-, Ca2+-, and cytosol-dependent steps in vesicle formation and fusion with the acceptor membranes [5], but the mechanism by which these vesicles are formed remains unknown. Logically, the initial step in the outgrowth of ER membrane and the assembly of transport vesicles should involve biosynthesis of their membranes and include the enzymes responsible for membrane growth and containment of the transported cargo. Since biosynthesis of the major membrane phospholipid, i.e., phosphatidylcholine (PC), is regulated by reversible translocation of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase from the inactive state in the cytosol to the endoplasmic reticulum, where it is activated [lo, 111, we investigated the role of this enzyme in the growth of transport vesicles and analyzed the catalytic activity of the coat protein assembled on the vesicles. Here we present evidence that the components of the vesicular coat display cytidylyltransferase and diacylglyceroltransferase enzymatic activities, which, when subjected to phosphorylation or fusion with Golgi membranes, dissociate from the vesicle surface.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de nouo synthesized phosphatidylcholine (PC) wasincorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 m&f N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Znz+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed. o 1992 Academic PESS, IN.

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be ad-

321

0014.4827192

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Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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buffered saline, pH 7.2, immediately following dissection and rinsed twice with the same buffer to remove mucus, which would interfere with subcellular fractionation. The rinsed tissue was homogenized in 2 vol of ice-cold 37.5 mM Tris-maleate buffer, pH 6.5, containing 0.5 M sucrose, 5 mM MgCl,, and 1% dextran with a Polytron 20ST for 45 s at 6000 rpm. Nuclei and intact cells were removed by centrifugation at 6000 rpm for 15 min, and the supernatant was removed, diluted with homogenization buffer to a volume of 10 ml/g tissue, and centrifuged at 10,OOOg for 20 min to yield a pellet consisting of mitochondria. The postmitochondrial supernatant was layered onto a discontinuous sucrose gradient consisting of 2.0, 1.5, and 1.3 h4 sucrose layers and centrifuged at 85,000 rpm for 90 min. The crude fraction of the intracellular membranes was removed from the top of the 1.25 M sucrose layer, adjusted to 1.20 M sucrose, layered over 2.0,1.6, and 1.4 M sucrose layers, overlaid with 1.0 and 0.5 M sucrose in 10 mM Hepes-KOH buffer, pH 7.4, and centrifuged for 2.5 h at 100,OOOg. Fifteen fractions were collected from the bottom of the tube; each fraction was mixed with 3 vol of Tris-maleate buffer, pelleted for 20 min at 7O,OOOg, and resuspended in 0.5 ml of the same buffer. Following the distribution and enrichment of the classical membrane enzyme markers, the ER membranes were collected from interface 2.01 1.6 M sucrose, whereas the Golgi membranes were collected from the 1.211.0 M sucrose interface. Immunodetection and quantitation of mucin precursors and integral proteins of ER and Golgi membrane preparation. Two hundred micrograms of the ER and Golgi membrane fractions were treated with 1% Triton X-100 and centrifuged, and the supernatant was adjusted with phosphate-buffered saline to 4 pglpl, coated in serial dilutions onto 96-well microtiter plate, and incubated at 4°C overnight. For the control, 250 ng of deglycosylated gastric mucus glycoprotein (apomutin) or 1 pg of intact mucus glycoprotein was coated in the same manner. After overnight blocking with 1% bovine serum albumin and normal goat serum, the anti-mucin monoclonal antibodies (culture medium from 3G12 clones) were added, incubated for 2 h at room temperature, and reacted with the diluted (1:lOOO) horseradish peroxidase (HRPD)-conjugated goat anti-mouse immunoglobulin for additional 2 h at room temperature. For the detection, o-phenyldiamine (0.4 mg/ml in 1.0 M citrate, 0.2 M sodium phosphate buffer, pH 5.0) containing 0.1% H202 was used. After 30 min, the reaction was terminated with 2 M H,SO, and the absorbance determined at 490 nm using a Titertek Multiscan Spectrophotometer. A portion of the solubilized samples was subjected to 7.5% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with the antibodies recognizing the integral ER proteins ((YRI MAb for ribophorin I and 2B7 MAb for protein fatty acyltransferase) [13,16]. The secretory protein (apomucin) was immunostained using antiapomucin 3G12 MAb [12]. In separate experiments, freshly prepared membranes were used as source of ceramide:UDPglucose glycosyltransferase activity, an exclusive marker of Golgi membranes [15]. Isolation of ER transport vesicles from gastric mucosul cells. Transport vesicles from rat gastric mucosal cells were prepared by a modification of the procedure described in [16]. The ER membranes were incubated in a mixture consisting of 0.1 mglml mucosal ER and 6 mg/ml of bovine brain cytosol prepared as described in [16] and enriched with 5 mM palmitic acid, 5 mM oleic acid, 15 mM CoA, 7.3 IU/ml creatine kinase, 2 mM creatine phosphate, 50 aM ATP, 50 PM CTP, 250 aM UTP, 25 mM Hepes-KOH (pH 7.0), 20 mM KCl, 0.2 M sucrose, 0.2 mM DTT, 20 PM GTP, and 50 rglml RNase. The mixture was assembled on ice and then incubated in the presence of [i4C]phosphocholine (0.005 &!ilpl) at 37°C for 30 min. Subsequently, the incubation mixture was cooled to 4°C and centrifuged through a 0.3 M sucrose layer at 100,OOOg for 20 min. The pellet was subjected twice to the treatment with 25 mM Hepes-KOH (pH 7.2), 250 mM KCl, 2.5 mM magnesium acetate, and 0.2 M sucrose (stripping buffer). Each time, after 15 min incubation on ice, the membranes were centrifuged at 100,OOOg for 20 min. The supernatants from the first and second stripping treatments were combined and pelleted at 150,OOOg for 1 h.

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Further purification of the transport vesicles was achieved on a discontinuous sucrose gradient consisting of 1 vol of 50% sucrose, 2 vol of 40% sucrose, 4 vol of 35% sucrose, and 0.5 vol of 30% sucrose, prepared in stripping buffer [16]. After centrifugation at 200,OOOg for 16 h, the gradient was collected into 20 fractions and the aliquots were subjected to SDS-PAGE immunoblotting, phospholipid detection and quantitation, and the enzyme activity assays. Determination of CTPphosphocholine cytidylyltrunsferuse and 1,2diucyl-sn-glycerol:CDP-choline phosphotrunsferuse activity. Twenty micrograms of ER membranes or 2 rg of ER transport vesicles were suspended in 25 ~1 buffer consisting of 25 pmol Tris-HCl, pH 6.5,0.35 pmol magnesium acetate, 12.5 nmol ATP, and 40 nmol dithiothreitol. Following 10 min preincubation, 25 ~1 of mixture containing 15 nmol of [i4C]phosphocholine (20 &ilhmol) and 20 nmol CTP were added [17]. For the assay of the phosphorylated dissociated cytidylyltransferase, the mixture was supplemented with phosphatidylcholineoleic acid liposomes. To detect 1,2-diacyl-sn-glycerol:CDP-cholinephosphocholine transferase activity 20 nmol of diacylglycerol was added and after 15 min at 37”C, the reaction was terminated by immersion in boiling water for 2 min [17, 181. Protein precipitate was removed by centrifugation at 10,OOOg for 10 min and the supernatant was extracted with 3 vol of chloroform/methanol (2/l, v/v). The water layer was applied to a Silica Gel 60 thin-layer plate (10 X 10 cm) and developed in methanol/0.6% NaCl/NH, (10/10/l, v/v/v). The organic extract of the incubation mixture was chromatographed in chloroform/methanol/water (60/35/8, v/v/v). The dried plates were subjected to radioactivity scanning using a Berthold LB 286 Digital Autoradiograph, or the bands corresponding to CDP-choline and PC were scraped into scintillation vials, mixed with 0.5 ml water and scintillation fluid, and counted. Incubation of ER transport vesicles under phosphoryluting and dephosphoryluting conditions and release of phospholipid-synthesizing enzymes. Ten micrograms of ER transport vesicles were incubated for 30 min at 37°C under phosphorylating conditions with 25 U of the pure CAMP-dependent protein kinase, 0.1 mM [y-“P]ATP, 4 mM MgCl,. The mixture was enriched with 250 nM okadaic acid and fractionated into supernatant and pellet by centrifugation at 130,OOOg for 3 h. The supernatant was passed through a Q-Sepharose column to separate the CAMP-dependent protein kinase and then subjected to cytidylyltransferase and phosphocholinetransferase activity determination in the presence of phosphatidylcboline-diacylglycerol-oleic acid liposomes, SDS-PAGE, and autoradiography [19]. The pellet consisting of ER transport vesicles depleted of phospholipid-synthesizing enzymes was used for fusion with Golgi directly or the vesicles were first subjected to depbosphorylation with 20 U of alkaline phosphatase and then used for fusion assay. To achieve dephosphorylation, the vesicles were incubated for 1 h at 4°C under continuous gentle mixing with 20 U of alkaline phosphatase bound to agarose. The alkaline phosphatase was removed by centrifugation at 2OOOg for 5 min and the supernatant was then centrifuged at 130,OOOg for 1 h to sediment the transport vesicles, which then were utilized for fusion experiments. In the experiments where ER transport vesicles were used for fusion with Golgi, [‘4C]phosphocholine was used for labeling the de nouo synthesized phospholipids in the vesicles while nonradioactive ATP was used in the phosphorylation assays. The recovery of the transport vesicles was estimated in terms of i4C counts, utilized, and recovered following phosphorylation and dephosphorylation treatments. Fusion of ER transport vesicles with Golgi. In this experiment, the donors, ER transport vesicles, were radiolabeled with [‘“Clphosphocholine (lO,OOO-15,000 cpm/lO pg vesicles). The vesicles used were without any treatment, or phosphorylated with CAMP protein kinase, or subjected to phosphorylation and dephosphorylation. The incubation mixtures consisted of 50 fig radiolabeled vesicles (SOOO-12,000 cpm/lO pg), 150 pg cytosol, 250 ng NSF (authentic NSF was obtained from Dr. Beckers, or the NSF-containing fraction was

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prepared in our laboratory), 33 mM Hepes-KOH, pH 7.0, 33 mM KCl, 2.5 mM magnesium acetate, 20 mMcreatine phosphate, 10 U/ml creatine phosphokinase, 0.7 mM ATP, 0.3 mM CTP, 0.3 mM GTP, 0.1 mM UTP, and loo-150 pg of Golgi membranes. After addition of Golgi membranes (acceptor), the transport-fusion experiment was initiated by the transfer of the mixture to a 37°C bath and incubation for 15-60 min. The reaction was terminated by transfer to ice; the samples were layered on a discontinuous sucrose gradient and subjected to centrifugation to separate the fraction of Golgi and unfused membranes [16]. The Golgi membranes, the recovered free vesicles, and the remaining intermediate fractions were analyzed for the content and composition of phospholipids labeled with [‘4C]phosphocholine precursor. The apomucin transferred from ER to Golgi was quantitated using 3Gl2 antiapomucin MAb in the indirect ELBA assay.

RESULTS

Characterization of ER and Golgi Preparations Gastric Mucosal Cells

from

To validate the suitability of ER and Golgi preparations from gastric epithelial cells in the reconstitution of the intracellular events leading to the synthesis of ER transport vesicles, we measured the intracellular transit of the mucus glycoprotein apopeptide. A characteristic feature of this 0-glycosidic glycoprotein elaborated in gastric epithelium is the presence of 60- to 65kDa apopeptide, which, when transferred to Golgi, undergoes extensive glycosylation, resulting in the attachment of multiple carbohydrate chains, each with GlcNAc(Gal)GalNAc-0-Ser(Thr)core [20]. Thus, to determine the apomucin translocation from ER vesicles to Golgi, the glycosylation with UDP [3H]galactose and immunoprecipitation (not shown) or immunodetection and quantitation of the transported substrate by ELISA assay were performed. Here, since Golgi of gastric mucosal cells was substituted with Golgi from liver (which is totally free of mucin; Fig. l), the estimate of the net transport-fusion by immunoquantitation of the mucin peptides within Golgi was used. Based on the ELISA assay, it was found that 1 pg of protein of ER transport vesicles contains 90 +- 20 ng apomucin, of which 20-35% is delivered to Golgi during 30 min fusion (4.0-7.3 ng apomucin/pg Golgi proteins). Characterization of the ER Transport Mucosal Cells

Vesicles of Gastric

The ER transport vesicles of gastric mucosal cells were generated from ER purified according to method described under Experimental Procedures. As illustrated in Fig. 2, the preparation was free of RER elements. The immunodetection of the integral protein of ER with antiribophorin I (YRI MAb and with 2B7 MAb against protein fatty acyltransferase showed that both components were enriched in ER preparations, but undetectable in the ER vesicles and Golgi preparations. In contrast, the ceramide:UDPglucose glucosyltransferase

FIG. 1. Immunodetection of the in vitro intracellular transport of apomucin from ER transport vesicles to Golgi. The reaction of gastric apomucin (25 ng, prepared by enzymatic deglycosylation of the mature mucin) with antimucin lH7 MAb (left) and 3G12 MAb (right) is shown in lane 1. Lanes 2-7 were developed with antimucin 3G12 MAb and represent, from left to right, the immunoreaction of 25,50,100 ng of Triton X-100 extract of ER membranes (lane 2), 3,6, 12 ng of ER transport vesicles (lane 3),3,6, 12 ng of transport vesicles treated with trypsin (1 mg/ml, 15 min at 37°C) to destroy the antigenicity of the apomucin which is not internalized (lane 4),0.3 and 0.5 fig of 1% Triton X-100 extract of the liver Golgi (lane 5),0.15,0.3,0.6 eg of the 1% Triton X-100 extract from Golgi of the liver following fusion with gastric ER transport vesicles (lane 6), and 0.3, 0.6 wg of the 1% Triton X-100 extract from Golgi of liver, which, prior to extraction, was subjected to trypsin digestion with 1 mg trypsin/ml for 15 min at 37°C (lane 7).

was detected only in Golgi membranes (Fig. 3). The electron microscopic study of the mucosal ER, after incubation generating ER transport vesicles (in the presence of GTP$S), revealed a population of sealed, dense granules of 80-100 nm in diameter, and occasionally small microsomes with vesicles in the process of detachment were observed (Fig. 4, lower right corner). Together, the nonidentity of ER transport vesicles with ER membranes or with Golgi, the containment of the apomucin cargo, and the EM appearance of the 80- to lOO-nm elements indicated that the isolated vesicles are not nonspecifically pinched off ER or Golgi fragments, but are instead the morphological elements carrying proteins from ER to Golgi. The results of the generation of vesicles in the presence of phospholipid precursor [14C]phosphocholine revealed that 80% of the incorporated label was in the vesicles and was contained in phosphatidylcholine (Fig. 5A). The synthesis of phosphoglycerides, transport vesicle formation, and CTP:phosphocholine cytidylyltransferase activity were all sensitive to N-ethylmaleimide (NEM) and Zn’+. In the presence of 3 mM NEM or

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FIG. 2. Western blot analysis of the endoplasmic reticulum (ER), transport vesicles (V), and Golgi (G) proteins with monoclonal antibodies against integral proteins of ER, with 2B7 MAb against protein fatty acyltransferase (left) and with aR1 MAb against ribophorin I (right). The delipidated protein extracts (5-10 pg each) were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with respective antibodies.

Zn2+, the process of vesicle formation and enzyme activity were both inhibited by 70-80% or completely arrested (Fig. 5). These data, together with the results showing 50% or more inhibition of the vesicle formation in the incubation system not supplemented with fatty acyl-CoA or

cts

cpm FIG. 3. cles (V), and used as the [3H]glucose and developed the Berthold

C

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depleted of choline, allowed us to speculate that the formation of ER transport vesicles is connected with the synthesis of phosphatidic acid and phosphatidylcholine and the activity of the enzymes involved in this process. To find out whether ER-derived transport vesicles contain the enzymes that were responsible for their membrane generation, the purified vesicles were incubated with precursors of phosphatidylcholine and the reaction products were analyzed. In the presence of [‘4C]phosphocholine and CTP, the vesicles and the dissociated coat proteins of the vesicles generated labeled CDP-choline (Fig. 6), whereas upon addition of diacylglycerol, [ 14C]phosphatidylcholine was synthesized (Fig. 7). This study revealed that the proteins that remain on the transport vesicles have catalytic activity of CTP:phosphocholine cytidylyltransferase and 1,2-diacylglycerol phosphocholine transferase. Phosphorylation

of Transport Vesicles

CTP:phosphocholine cytidylyltransferase is the ratelimiting enzyme in the synthesis of phosphatidylcholine and exists in two interconvertible forms: the phosphorylated inactive cytosolic form and the ER-bound dephosphorylated active form [21]. Since phosphorylation of the enzyme stimulates its dissociation from the membrane, experiments were carried out to determine whether cytidylyltransferase activity of the coat pro-

V 2,496

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Identification of the ceramide:UDPglucose glucosyltransferase activity in preparations of Golgi membranes (G), transport vesiendoplasmic reticulum (ER) and isolation of the glucosylceramide products. The membrane proteins (50 and 100 jig from each) enzyme source and the control (C!) containing 100 pg of boiled Golgi membranes were incubated with the ceramide and UDP under conditions described in [20]. The chloroform extracts of the incubation mixture (800 pl each) were applied to a HPTLC plate in a solvent system consisting of chloroform/methanol/water (60/35/B, v/v/v) and the amount of [3H]Glc-Cer quantitated in LB 286 Digital Autoradiograph. The net counts (counts1100 min) and cpm for each sample are shown below respective lanes.

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from the vesicles during phosphorylation consist of at least two enzymes involved in the synthesis of phosphatidylcholine (CDP-choline pathway) with the possibility of two other proteins being involved in the synthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. The speculation concerning phosphatidylethanolamine synthesis is based on results obtained when the ER-transport vesicles were generated in the presence of 3H-labeled fatty acids and the isolated vesicles contained radiolabeled phosphatidylethanolamine (unpublished results). The formation of labeled phosphatidylethanolamine suggested that similar to phosphatidylcholine, the enzymes of the CDP-ethanolamine pathway may be present on the vesicles or that demethylation of phosphatidylcholine is taking place. Although the latter process must not be dismissed, the data presented by Vance and Vance and Samborski et al. [22,23] provide evidence that in ER the CDP-ethanolamine pathway plays the leading role in phosphatidylethanolamine generation. Thus, it is tempting to suggest that these enzymes constitute the dissociating proteins of the ER transport vesicles. Phosphorylation, Dephosphorylation of ER Transport Vesicles, and Their Potential to Fuse with Golgi Membranes

FIG. 4. Electron micrograph of the preparation from rat gastric mucosal cells. (Top) The preparation of ER before the incubation that generates ER transport vesicles (X10,000). (Bottom, upper right corner inset) ER membranes after incubation (X75,000). The separated vesicles (v) and the vesicles in the process of separation (inset) are distinguished here. The average size of the ER transport vesicles found was in the range of 80-100 nm in diameter. However, larger formations with dense contents (V) were also observed.

teins of the transport vesicles is affected by phosphorylation. As shown in Fig. 8, incubation of the transport vesicles with [T-~~P]ATP and the catalytic subunit of CAMP-dependent protein kinase resulted in the phosphorylation and dissociation of at least four proteins, one of which displayed activity of CTP:phosphocholine cytidylyltransferase and generated CDP+holine (Fig. 7). The other proteins were also catalytically active in the synthesis of phospholipids. As shown in Fig. 7, the incubation of the dissociated proteins with [14C]phosphocholine, CTP, and diacylglycerol afforded radiolabeled products identified as phosphatidylcholine. These results provide evidence that the proteins dissociating

In other cells, decoating of the transport vesicles is a prerequisite to the initiation of the next stage of transport [l, 3, 161. Thus, it is possible that release of the vesicle-bound enzymes (which are involved in the synthesis of phospholipids) is in part equivalent to decoating and to induction of the fusion with Golgi. As shown in Fig. 9, the vesicles subjected to phosphorylation and dephosphorylation were 30-40% more active in fusion with Golgi. However, if the entire incubation mixture was subjected to phosphorylation, fusion proceeded with only 10% efficiency. Apparently, some dephosphorylated proteins are required for specific recognition between vesicle and Golgi membrane or for regulation of the transport between ER and Golgi. DISCUSSION The development of in vitro assays that reconstitute transport from ER to Golgi and the methodology for isolation of transport vesicles afforded significant progress in understanding the chronological progression of the reactions and their N-ethylmaieimide, ATP, and Ca2+ sensitivity [4]. In yeast, a large number of proteins have been recognized to be the required components of intracellular transport from ER to cell surface [24-261. The ER to Golgi transport-supporting proteins include cytosol and membrane-bound factors, which induce growth and budding of transport vesicles independently

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net (CW

Vl v2 v3 ER4 ER5 ER6

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46,190 8,933 4,225 39,847 8,344 4,188

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1,925 375 175 1,494 315 158

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FIG. 5A. Incorporation of [i4C]choline into phosphatidylcholine of the ER transport vesicles (V) under optimal conditions (Vl, ER4) and in the presence of 3 mM NEM (VZ, ER5) and 3 mM Zn *+ incubation contained 10 mg of ER membranes and consisted of components detailed under Experimental the vesicles and the residual ER membranes were recovered by differential centrifugation; each sample chloroform/methanol (2/l, v/v), HPTLC m a solvent system consisting of chloroform/methanol/water a Berthold LB 286 Digital Autoradiograph.

from the components conferring ER stability. This links these components to protein vesicular transport, but how the transporting elements are formed and

FIG. 5B. Immunoquantitation of the apomucin transported by the vesicles prepared as described in the legend to Fig. 5A. Gastric apomucin was immunoprecipitated with 3G12 MAb from lipid-extracted protein residues of the vesicles and of the residual ER membranes and subjected to 10% SDS-PAGE and silver staining. Apo, rat gastric apomucin standard; ER, apomucin precipitated from ER4 residual membranes; PM, prestained markers consisting of 130-, 75-, and 50-kDa peptides; Vl, V2, V3, apomucin recovered from the transport vesicles obtained under optimal conditions (Vl) and in the presence of NEM (V2) and Zn2+ (V3).

and the residual ER membranes (ER) (V3 , ER6). Each sample subjected to Procedures. Following incubation, was subjected to lipid extraction with (80/35/5, v/v/v), and radioscanning in

which proteins and factors are responsible for their elaboration are still unknown. Obviously, such proteins should be involved in biogenesis of the membrane and in recognition of the Golgi membrane. For the first role, the best candidates are lipid-synthesizing enzymes. The enzymes involved in the synthesis of cholesterol, many of which are located in endoplasmic reticulum, should be dismissed, considering that cholesterol is transported from ER to plasma membranes in separate vesicles which were found to bypass the Golgi [27]. The other possibility to consider is the involvement of the enzyme-synthesizing phospholipids, particularly phosphatidylcholine and phosphatidylethanolamine, which are the major contributors in building the membranes. Our studies with [14C]phosphocholine precursor, which for incorporation into phosphatidylcholine requires intervention of two enzymes (CTP:phosphocholine cytidylyltransferase and 1,2diacylglycerol:CDPcholine phosphotransferase), showed that over 80% of the label was recovered in phosphatidylcholine of the newly formed ER transport vesicles and 1520% remained in the phosphatidylcholine of the ER residual fraction after “stripping” of the transport vesicles. This distribution probably reflected the partially completed transport compartments that were not ready for separation, since additional incubation of the residual ER with unlabeled PC precursor chased about 50% of the label to the subsequent batch of vesicles. Moreover, the par-

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FIG. 6. Enzymatic activity of the proteins retained on ER transport vesicles. Detection of CTP:phosphocholine cytidylyltransferase activity. The ER transport vesicles of rat gastric mucosal cells were incubated with [‘4C]phosphocholine and the water-soluble products were identified following HPTLC in methanol/0.6% NaCl/NH, (101 10/l, v/v/v) and autoradiography. 1, [Wlcholine; 2, CDP [‘“Clcholine; 3, [‘4C]phosphocholine; 4, products derived from incubation of ER transport vesicles with [‘4C]phosphocholine and CTP, 5, control consisting of boiled transport vesicles (3 min), [‘“Clphosphocholine, and CTP.

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tial NEM sensitivity of the vesicular transport is also reflected in the detrimental action of NEM on the CTP:phosphocholine cytidylyltransferase. In the presence of 3 mM NEM, the formation of the transport vesicles is reduced to lo-14% of the control value, while the activity of the enzyme is decreased from 3.7 nmol/min/ mg to 0.17 nmol/min/mg, a drop of 90-95%. In summary, the experiments performed to produce transport vesicles, together with the studies on CTP:phosphocholine cytidylyltransferase enzyme activity and its general properties, suggest that the NEM sensitivity and ATP and fatty acyl-CoA factors are affecting steps in the process of the vesicle assembly that are even earlier than suggested, namely, the formation of vesicular membrane. The studies with semi-intact cells imply that the transport rate-limiting factor dissociates after initiation of transport [4]. Similarly, the rate-limiting enzyme of membrane phosphoglyceride synthesis, CTP:phosphocholine cytidylyltransferase, is transiently released to cytosol [al]. Therefore, it is tempting to speculate that the limiting factors of semi-intact cell transport [4] and the components of the coat proteins observed by others [32] include the phospholipid-synthesizing enzymes.

A

tially attached or incompletely separated transport vesicles from the microsomal membranes were also observed in electron microscopic studies (Fig. 4). The results thus indicate that de novo synthesized phosphatidylcholine is used for the generation of the new membranes that become the transport vesicles and that CTP:phosphocholine cytidylyltransferase and 1,2-diacylglycerol:CDP-choline phosphotransferase, together with other factors, are responsible for this process. Therefore, the requirements and functional properties of these enzymes, together with conditions and factors described [4], dictate that the incubation of ER membranes generating transport vesicles be conducted in the presence of cytosol to provide a source of choline, choline kinase, glyceryl phosphate, fatty acyl-CoA, ATP, and CTP [ll, 18, 281. It was speculated that fatty acyl-CoA might be necessary for post-translational modification of proteins involved in the transport [29]. Undoubtedly, the function of many proteins is affected by acylation, but in keeping with our findings, fatty acylCoA appears to be required the most for the synthesis of diacylglycerolphosphate (phosphatidic acid). Earlier studies showed that when membranes are prepared with NEM, their ability to transport is lost, but can be restored by the addition of cytosol [I, 3, 41. This transport restorative property of cytosol was believed to be directly dependent on the presence of NSF component [16,30, 311. In light of our findings, the ini-

B

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FIG. ‘7. Enzymatic activity of the proteins dissociating from ER transport vesicles. The proteins dissociated from the ER transport vesicles and subjected to CAMP protein kinase dependent phosphorylation were obtained and incubated with [i4C]phosphocholine, CTP, and diacylglycerol as detailed in Experimental Procedures, and the aqueous and the organic phase of the incubates were subjected to thin-layer chromatography and autoradiography. (A) Phosphatidylcholine (upper spot) and sphingomyelin (lower spot) standards separated by two-dimensional chromatography in solvent systems: chloroform/methanol/water (65/25/4, v/v/v) (vertical) and l-butanol/acetic acid/water (61212, v/v/v) (horizontal). (B) Organic extract of the incubate chromatographed in the solvent mixture chloroform/methanol/water (65/25/4, v/v/v). The position of the radiolabeled lipid corresponds to the phosphatidylcholine migration. (C!) The HPTLC separation of the water-soluble products of the incubation mixture in methanol/0.6% NaCl/NH, (10/10/l, v/v/v). The CDP [“Clcholine product (identified by an asterisk) appears above the [‘*Clphosphocholine substrate.

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SLOMIANY

FIG. 8. (Right) SDS-PAGE (5%) separation of the proteins dissociating from ER transport vesicles following incubation with [ya’P]ATP and protein kinase (lane 2) or released with 0.1 M sodium carbonate, pH 11 (lane 3). Lane 1 represents the total proteins of ER transport vesicles (prepared in the presence of GTPrS), and lane 4 represents the total proteins of the endoplasmic reticulum. The dissociated and 32P-phosphorylated proteins of the ER transport vesicles are depicted (left) (8% SDS-PAGE): S, the proteins that dissociated from the vesicles; V, proteins that remained on the vesicles. The gel was exposed to X-ray film for 3 h at -70°C.

Several lines of evidence support this contention. Analyses of the vesicles have shown enzymatic activity characteristic of CTP:phosphocholine cytidylyltransferase and CDP-choline:diacylglycerol phosphotransferase. The enzymes dissociate from the vesicular membranes and the process is potentiated when the vesicles are subjected to phosphorylation. The released proteins retain their enzymatic activity, and when incubated with phosphocholine, diacylglycerol, and CTP, phosphatidylcholine is synthesized. SDS-PAGE and autoradiography indicate that phosphorylation of the transport vesicles causes more than the dissociation of the proteins that qualify as cytidylyltransferases or other known phosphoglyceride-synthesizing enzymes [ 18,211. A variety of possibilities for temporary occupancy by other proteins exist, including those that are essential for shaping, pinching off vesicles, and recognizing fusion sites, or those that are directly involved in the fusion itself. The multiplicity of the components detected on these transport vesicles in contrast to those generated in Golgi suggests that these vesicles come to exist by the action of numerous enzymes and factors, whereas the transport vesicles formed by subsequent organelles require only some specific modifications or attenuation to their new function. It is possible that this type of vesicle is used to deliver some of the protein product to the site of their modification and to remodel and renew the membranes, whereas the other intracellular transfers are achieved by means of membrane tubules or vesicles that could shuttle between organelles and replace the lost membranes during

ET

AL.

enterograde transport [33]. In our experimental design, it was not possible to assess the other processes or the magnitude of the retrograde transport, since the unlabeled Golgi membranes had to be used for detection of enterograde transport and membrane fusion. Thus, the retrograde transfer, if any, contained mostly unlabeled lipids and hence could not be measured. Also, we could not quantitate the flow of phospholipids by means of phospholipid transfer proteins [34]. In our opinion, however, the latter mode of compensation against the tide of phospholipids, moving as protein-packed vesicles, is more appealing than vesicular shuttle. One strong argument against vesicular shuttle is that this process would provide ER membranes with glycosphingolipids (occupying the luminal surface of Golgi membranes),

300( Cl

Control

n

Decoated

fit

Phorphorylaled

vesicles vesicler

200(

0) a E : 2 u” 1ooc

C

FIG. 9. The phosphorylation and dephosphorylation effect of ER transport vesicles on their fusion potential with Golgi. In each experiment, Golgi membranes were preincubated with 1 mM NEM at 37°C for 15 min followed by addition of dithiothreitol to 2 m&f. The cytosol was prepared as described under Experimental Procedures except in experiment 4, in which the cytosol and ER vesicles were subjected to phosphorylation (25 U of CAMP-dependent protein kinase, 0.1 mM [T-~*P]ATP, 4 md4 MgCI, for 30 min at 37°C). The ER transport vesicles were treated as follows: 1, control, fusion under optimal conditions; 2, decoated vesicles that were subjected to phosphorylation as described above and dephosphorylation (20 U of alkaline phosphatase bound to agarose for 1 h at 4°C under continuous gentle mixing); 3, decoated vesicles that were subjected to phosphory lation only and reisolated; and 4, vesicles and cytosol subjected to phosphorylation only. In all experiments, the fusion potential is expressed in the amount of [“Clcholine-labeled phospholipids incorporated into Golgi membranes.

PC

whereas transport proteins with the cytosolic domain neither glycosphingolipids moved during this type of to ER.

SYNTHESIS

AND

ER

exchange their lipid cargo of the membrane, and thus nor sphingomyelin are reflow of phospholipids back

We are very grateful to Dr. G. Kreibich, polyclonal and monoclonal antiribophorin C. Beckers, Sloan-Kettering Institute, for anti-NSF antibody, and Mr. F. Macaluso microscopy studies. This research was AA05858-10 from NIAAA and DK21684-15

Rockefeller University, for I antibodies. We thank Dr. his gift of NSF protein and for performing the electron supported by-NIH Grants from NIDDKD.

VESICLE

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