Prechylomicron transport vesicle: isolation and partial characterization

N. Suresh Kumar and C. M. Mansbach II

Department of Medicine, Division of Gastroenterology University of Tennessee, Memphis 38163; and Veterans Affairs Medical Center, Memphis, Tennessee 38104


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The intestine is able to regulate its output rate of chylomicrons, the major intestinal triacylglycerol (TG) transport vehicle. We have proposed that a vesicle, transporting the developing chylomicron from the endoplasmic reticulum (ER) to the Golgi, is the rate-limiting step in the process of TG transit through the enterocyte [Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G18-G30, 1997]. We wished to isolate and characterize this vesicle. The apical portion of rat intestinal cells were avulsed, and the mucosa was stirred in buffer. The supernatant was centrifuged in two different sucrose gradients, and the top 2.5 ml of the last gradient were collected and concentrated. Electron microscopy showed a 200-nm vesicle. The vesicle contained immunoidentifiable apolipoprotein (apo) B48 and apo A-IV but very little apo A-I, although apo A-I was present in the ER and Golgi. [3H]TG-loaded vesicles delivered [3H]TG to the Golgi but not the ER. Marker enzyme assays also indicate that the isolated fraction is different from the ER and Golgi fractions. We conclude that we have isolated a vesicle that is post-ER but pre-Golgi that vectorially transports TG to the Golgi.

vesicles; chylomicrons; intestinal lipid transport; lipid absorption; triacylglycerol


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

INTESTINAL ABSORPTION of fat and its subsequent secretion from the enterocyte as chylomicrons is a complex cellular event, involving the coordinate synthesis of apolipoproteins and lipids, and their intracellular assembly into mature lipid-containing particles. The chylomicron, the unique triacylglycerol (TG)-rich lipoprotein of the intestine, is a heterogeneous globular particle, 100-500 nm in diameter, whose lipid composition consists mainly of TG with some cholesterol, cholesteryl esters, and phospholipids. The surface of the chylomicron contains both exchangeable apolipoproteins (apo) A-I, apo A-IV, apo C, and apo E and the nonexchangeable apo B48 (7). The overall throughput of TG by the intestine is physiologically regulatable and is highly efficient provided enough phosphatidylcholine is present (18, 23, 33).

The process of assembly of the TG portion of the chylomicron begins with the rapid re-esterification of absorbed monoacylglycerol (MG) and fatty acids to TG, which occurs at the level of the endoplasmic reticulum (ER) (16). TG synthesis occurs on the cytoplasmic face of the ER, and as a consequence the TG must translocate to the ER lumen as a prerequisite for its transepithelial secretion. It was originally thought that the newly synthesized TG was added to the growing chylomicron particle by the microsomal triglyceride transfer protein (MTP) (38), but more recent data suggest a greater role for MTP in translocating apo B across the ER membrane (6). In either event MTP plays a crucial role in the secretion of lipid-rich lipoproteins because in its absence no lipid-rich lipoproteins are secreted from either the intestine or liver (8).

The developing chylomicron must be transported from the ER to the Golgi before its basolateral secretion (19). This step has been proposed to be rate limiting in terms of TG export by the intestine (15). Several pieces of evidence support this hypothesis. Lipid droplets have been shown to accumulate in the ER awaiting transport to the Golgi in both normal and pathophysiological conditions (1, 5). Pluronic L-81, a nonionic detergent that severely reduces lymphatic chylomicron output, blocks the intracellular movement of TG at the level of the ER (35). In the rare genetic disorder, chylomicron retention disease, TG is shown to accumulate in the ER, suggesting a transport block in the secretory pathway (22). The affected individuals are unable to secrete chylomicrons and malabsorb lipid despite normal synthetic rates of apo B synthesis and apo B mRNA editing (25).

Despite the wealth of information available on apolipoprotein synthesis and translocation across the ER membrane, the mechanism of its transport from the ER to the Golgi in lipoproteins is not clearly understood. It is hypothesized that the transport of macromolecules from one compartment to the other occurs via transport vesicles that fuse with specific acceptor membranes (24). One possibility for intracellular chylomicron movement is the vesicular transport system used by secretory proteins (31), as suggested by the sensitivity of TG export into the lymph by brefeldin A, a fungal metabolite that reversibly collapses the Golgi into the ER (19) and by the reduction in TG movement from the ER to the Golgi by N-ethylmaleimide treatment (15). However, the secretory protein paradigm is not the likely mechanism for chylomicrons in the intestine because we have shown (15) that TG movement from the ER to the Golgi is GTP independent, guanosine 5'-O-(3-thiotriphosphate) does not inhibit it, and antibodies to rab1 and rab2 also do not diminish TG movement to the Golgi.

In liver, although phospholipids and cholesterol could be shown to go from the ER to the Golgi by ATP and cytosolic-dependent processes similar to the vesicular transport for proteins, TG could not (21). In other studies, cholesterol-containing vesicles have been shown to bypass the Golgi apparatus completely and go directly to the plasma membrane, whereas vesicular stomatitis virus G protein containing vesicles follow the classical secretory protein vesicular transport system (36). In contrast to the vesicular movement of protein and lipid from the ER to the Golgi, fluorescent-tagged viral glycoprotein ts045 (vesicular stometitus virus glycoprotein-green fluorescent protein, VSVG-GFP) is transported from the ER in COS cells in many differently shaped, rapidly forming pre-Golgi structures, which are translocated to the Golgi along microtubules (27). Although microtubules have been proposed to traffic TG to the Golgi in the intestine (21), this is unlikely in view of our previous work showing that Golgi from donor rats was an effective acceptor for TG from the ER (15) and our unreported findings that colchicine does not inhibit the movement of TG to the Golgi (unreported observations).

To begin to resolve the issue of lipoprotein-TG movement from the ER, its site of synthesis, to the Golgi, we developed a cell-free system in which TG and apo B48 were actively transported from the ER to the lumen of the Golgi (15). The transport of TG (and apo B48) to the Golgi was shown to be vectorial in nature, to require ATP, a cytosolic protein, incubation at 37°C, and required intestinal Golgi as the acceptor. We proposed the mechanism by which this occurred to be vesicular in nature. To further our studies of this process, we first needed to isolate and characterize the putative vesicles. We report here the isolation and partial characterization of a functional vesicular fraction that vectorially transfers TG from the ER to the Golgi of the rat small intestine.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials. All biochemicals were purchased from Sigma Chemical (St. Louis, MO) unless noted otherwise. [Oleoyl-3H]triolein (TO, specific activity 28 Ci/mmol) was purchased from Dupont-New England Nuclear (Boston, MA). Antibodies to rat apo B48, apo A-I, and apo A-IV were generous gifts from Dr. Patrick Tso (University of Cincinnati, Cincinnati, OH). Zetaprobe membranes were obtained from Bio-Rad (Hercules, CA). All other reagents were the highest purity available from common vendors.

Isolation of putative vesicular fraction from rat small intestine. Male Sprague-Dawley rats (250-350 g) were maintained on Purina Rat Chow (Ralston Purina, St. Louis, MO) until used in the study. The rats were cannulated intraduodenally with PE-50 tubing (Clay Adams, Parsippany, NJ) and were infused through the cannula with 0.15 M NaCl, 5.37 mM KCl, and 5% glucose at 3 ml/h (Harvard infusion pump model 22; Harvard Apparatus, Millis, MA) overnight. The next day the rats were infused intraduodenally with a sonified emulsion of TO (in mM, 30 TO, 10 taurocholate, 10 Tris · HCl, pH 7.4) supplemented with [3H]TO (250 µCi) for 2 h at 4.5 ml/h. At the end of the 2-h infusion, 50 mg pentobarbital were given intraperitoneally and the proximal intestine was removed. The lumen was flushed with 10 mM taurocholate and then with ice-cold NaCl (150 mM). The intestine was filled with 150 mM NaCl solution containing 1 mM NaN3 and incubated for 5 min at 37°C. After the incubation, the intestinal segment was cut into smaller pieces (~8 cm), slit open, and the mucosal cell apical membranes were peeled off using a Zetaprobe membrane (Bio-Rad). The remaining mucosa was then placed in sucrose buffer (0.25 M sucrose, 10 mM HEPES, 0.01% NaN3, 2 mM phenylmethylsulfonyl fluoride, pH 7.3). The mucosa was gently agitated by swirling in the buffer that was subsequently centrifuged for 5 min at 1,000 g at 4°C to remove any whole cells released from the basement membrane. The supernatant was centrifuged at 40,000 rpm for 45 min in a 60-Ti rotor in a L8 M centrifuge at 4°C (Beckman Instruments, Fullerton, CA).

The resulting pellet was resuspended in the same buffer and 1.5 ml overlaid on top of a sucrose-step gradient consisting of 4.5 ml, 1.15 M, and 4.5 ml 0.86 M sucrose in 10 mM HEPES buffer, pH 7.3. This gradient was centrifuged for 9.84 × 106 g · min at 4°C in a SW-41 rotor. The 0.25/0.86 M interface was collected and diluted with 10 mM HEPES to 0.1 M sucrose as judged by its refractive index (Abbe refractometer) and overlaid on a continuous sucrose gradient (0.1-1.15 M sucrose in 10 mM HEPES, pH 7.3). The gradient was centrifuged for 7.38 × 106 g · min at 4°C, and 0.5 ml fractions were collected from the gradient by upward displacement with 1.15 M sucrose using an ISCO density gradient fractionator (model 640; ISCO, Lincoln, NE). Fractions containing [3H]TG were identified by differential extraction of 0.1 ml of each fraction (3) and determining its radioactivity in a liquid scintillation spectrometer (model 1500; Packard Instrument, Downers Grove, IL).

Isolation and subcellular fractionation of intestinal cells. The ER and Golgi fractions of isolated enterocytes were separated from the whole microsomal fraction of the proximal rat intestine using a sucrose-step density gradient as we described previously (15). "Heavy" and "light" Golgi are defined as the membranes floating at the 1.15/0.86 M and 0.86/0.25 M sucrose interfaces, respectively. The luminal contents of each subcellular fraction was released using 100 mM carbonate as described previously (13, 15).

Transport of [3H]TG from vesicles to ER and Golgi. The ability of vesicle-containing fractions to transport [3H]TG to the ER and Golgi was carried out by incubating the radiolabeled vesicular fraction (100 µg protein) with nonradiolabeled ER or Golgi (250 µg protein) in buffer (30 mM HEPES, 2.5 mM Mg acetate, 30 mM KCl, pH 7.3) containing an ATP-regenerating system [final concentrations, 1 mM ATP, 5 mM phosphocreatine, 5 units creatine phosphokinase, 1 mM CaCl2, 0.5 mM dithiothreitol (DTT) in 10 mM HEPES, pH 7.2] and 200 µg cytosolic protein at 37°C for 30 min. The suspension was then loaded onto a continuous sucrose gradient (0.1-1.15 M sucrose, 10 mM HEPES, pH 7.3) and centrifuged for 7.38 × 106 g · min at 4°C. The gradient was fractionated by upward displacement as above, and the [3H]TG radioactivity associated with each fraction was measured after its differential extraction (3) using a liquid scintillation spectrometer.

Preparation of cytosol. Cytosol used in the transport assay was prepared from the proximal one-half of rat intestine as described previously in detail (15). Briefly, intestinal mucosal cell preparations were homogenized in 0.25 M sucrose, 10 mM HEPES, and 5 mM EDTA, pH 7.3, and centrifuged at 13,500 rpm for 10 min. The postmitochondrial supernatant was then centrifuged at 40,000 rpm for 95 min in a 60-Ti rotor. The supernatant was collected and concentrated to 10 mg protein/ml by ultrafiltration using YM-10 DIAFLO membrane (Amicon, Beverly, MA). The concentrated cytosolic fraction was snap frozen in small volumes and stored at -70°C until used.

Detection of apolipoproteins. Vesicle proteins were subjected to SDS-PAGE on 4-20% gradient gels according to Laemmli and were electroblotted onto Zetaprobe membranes. The buffer system contained no methanol. Apo B48, apo A-I, and apo A-IV were detected using antibodies raised against rat apo A-I, apo A-IV and apo B48 in goats (the antibodies were a generous gift of Dr. Patrick Tso, University of Cincinnati); horseradish peroxidase-conjugated anti-goat IgG was used to detect the apoproteins. 3,3'-Diaminobenzidine tetrahydrochloride was used as the substrate for color development. The immunoblots were digitized and analyzed with the National Institutes of Health (NIH) image analyzer (NIH Image, version 1.61). The results are expressed in arbitrary densitometry units. Differences between means (n = 3) were analyzed by ANOVA using Bonferroni posttest multiple comparisons.

Enzyme assays. Marker enzymes were used to identify the purity of the various subcellular fractions studied and their activities compared with the whole homogenate. NADPH-cytochrome C reductase (17), acid phosphatase (8), succinate dehydrogenase (26), and galactosyl transferase (12) were used to identify the ER, lysosomes, mitochondria, and Golgi, respectively.

Two-dimensional PAGE of vesicle or membrane proteins. Membrane pellets were prepared by centrifuging the previously isolated subcellular fractions (ER, Golgi, or vesicles) at 16,500 rpm in an adapted SS-34 rotor in a RC-5C centrifuge (DuPont Sorvall, Wilmington, DE) at 4°C for 30 min. The protocol used was based on that employed by ESA (Chelmsford, MA) as described in their manual for two-dimensional (2-D) PAGE (Investigator 2-D Electrophoresis System Operating and Maintenance Manual). Briefly, 100 µg protein of each membrane fraction were suspended in 20 µl of boiling sample buffer A (0.3% SDS, 200 mM DTT, 28 mM Tris · HCl, 22 mM Tris base, pH 8), which was heated in a boiling water bath for 5 min. The suspension was cooled to room temperature, and 80 µl of sample buffer B (9.9 M urea, 4% NP-40, 2.2% of bio-lyte 3/10-100, 50 mM DTT, 0.01% bromphenol blue) were added. The suspension was incubated at 37°C for 30 min and was clarified by centrifugation for 2 min in a microcentrifuge at top speed. The supernatant (50 µl) was applied to isoelectric focusing (IEF) gels, which were cast in glass tubes (1.0 × 5 × 180 mm, Bio-Rad) using capillary action. IEF was performed for 17.5 h at 700 volts. Sodium hydroxide (100 mM) and phosphoric acid (10 mM) were used as the upper and lower electrode buffers, respectively. The IEF gel was extruded from the glass tubes using a plastic syringe filled with water. The gel was then equilibrated in equilibration buffer (0.3 M Tris base, 0.075 M Tris · HCl, 3% SDS, 50 mM DTT, and 0.01% bromphenol blue) for 2 min before carrying out the second dimension electrophoresis on 10% acrylamide using Laemmli's buffer system. After the second dimension run, the slab gel was fixed in 50% methanol and silver stained. All solutions used were filtered through 0.45-µm filters.

Electron microscopy. One drop of 20-fold concentrated vesicle suspension from the continuous sucrose gradient (fractions 1-5, Fig. 1) was allowed to air dry on an electron microscopy grid. The grid was stained with phosphotungstic acid and examined by electron microscopy as previously described (19).


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Isolation and identification of TG-containing vesicles. To isolate the prechylomicron transport vesicles (PCTV), a method different from the usual homogenization techniques to disrupt cells was required because this method resulted in an excess of ER and Golgi, which rendered attempts at isolating the PCTV by differential densities useless. Therefore, we utilized a technique that avulsed the apical membranes of cells (2, 14), leaving the ER and Golgi intact and allowing us to release the PCTV by swirling the remaining mucosa in buffer. The putative PCTV were purified first on a sucrose-step gradient and subsequently on a continuous sucrose gradient. The distribution of [3H]TG in the continuous gradient is shown in Fig. 1. Note that the [3H]TG appears toward the top of the gradient but not floating as would be likely if it were free TG. Although no attempt was made to quantitate the total recovery of vesicles, of the total dpm in the cellular homogenate ~0.33% was in the vesicular fraction in multiple trials. To determine where the ER and Golgi would distribute in the gradient, additional experiments were performed in which ER and light and heavy Golgi containing [3H]TG were isolated (15) from other rats infused intraduodenally with [3H]TO and separately centrifuged on the continuous sucrose gradient. As shown in Fig. 1, which is a composite of the four separate gradients, both ER- and Golgi-associated [3H]TG were more dense than the presumed PCTV-[3H]TG, thus suggesting that the [3H]TG appearing in the less dense region of the gradient was in a structure different from freely floating TG or chylomicrons and from the ER and Golgi.


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Fig. 1.   Distribution of [3H]triacylglycerol (TG) in continuous sucrose-density gradient. Figure is composite of 4 separate experiments in which prechylomicron transport vesicles (PCTV), heavy and light Golgi, and endoplasmic reticulum (ER) containing [3H]TG were separately placed on continuous sucrose-density gradient (0.1-1.15 M sucrose). Gradient was centrifuged, and fractions (0.5 ml each) were collected by pumping 1.15 M sucrose into bottom of centrifuge tube and collecting fractions from top. Thus lower-numbered fractions are the least and higher-numbered ones the most dense. Data show that each organelle or vesicle separately distributes in the gradient and that the PCTV are in the least-dense region.

To establish other differences between the PCTV and subcellular organelles besides density, we tested whether the PCTV expressed various enzyme activities known to be associated with specific organelles. The results are shown in Table 1. As shown in the table, no succinic acid dehydrogenase or galactosyl transferase activity was found in the PCTV. These enzymes are associated with the mitochondria and Golgi, respectively. The PCTV did express NADPH cytochrome c reductase and acid phosphatase activities which are associated with the ER and lysosomes, respectively. There was no increase in activity in either case over that found in the whole homogenate, indicating that there was no purification of the enzymes on subcellular fractionation. In contrast, NADPH cytochrome c reductase specific activity was eightfold higher in the ER compared with the whole homogenate as expected. Thus the enzymatic constituents of the PCTV are distinct from other subcellular organelles, including the recently described mitochondria-associated membrane (32).

                              
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Table 1.   Enzyme activity in subcellular compartments

To determine whether the isolated fraction from the top of the gradient contained vesicles, concentrated material was fixed and stained with phosphotungstic acid and examined at electron microscopy. Smooth vesicles were found in the isolated fraction. A typical structure of a vesicle (of 30 examined) is shown in Fig. 2. The diameter of these particles ranged from 100 to 200 nm but were mostly 200 nm in size.


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Fig. 2.   TG-containing vesicle, presumably developing chylomicron within vesicle, with small amount of collapsed membrane. Fluid from first 5 fractions of continuous sucrose-density gradient was concentrated, and 1 drop was placed on electron microscopy grid, dried, and stained with phosphotungstic acid (MATERIALS AND METHODS). Shown in figure is a membrane that surrounds lipid-containing material. Figure is representative of 30 vesicles examined.

Vectorial transport of TG from vesicles to Golgi. We reasoned that if the vesicle we isolated was the PCTV, it should deliver TG to the Golgi in a vectorial manner (15). To this end we prepared radiolabeled vesicles isolated from the low-density region of the continuous sucrose gradient by isolating them from donor rats that had been infused intraduodenally with [3H]TO. These 3H-labeled vesicles were incubated separately with both light and heavy Golgi and ER fractions prepared from rats that were naive to radioactivity at 37°C for 30 min under conditions in which transport of TG from the ER to the Golgi would be expected (15). After the incubation, each membrane fraction was separately placed on a continuous sucrose density gradient (see MATERIALS AND METHODS) to separate the vesicles from the ER and the two Golgi fractions which are more dense than the vesicles (Fig. 1). The results are shown in Table 2. When the vesicles were incubated either alone or with the ER, the [3H]TG remained primarily in the area of the gradient compatible with the PCTV. By contrast, when the vesicles were incubated with the heavy Golgi, 31% of the total dpm were displaced to the region of the gradient compatible with the light and 23% to the region occupied by the heavy Golgi. Thus a total of 54% of the available dpm were displaced to the Golgi region of the gradient. When the vesicles were incubated with the light Golgi, 33% of the 3H-dpm were displaced to the region of the light Golgi but only 5% to the region of the heavy Golgi. Few 3H-dpm from the vesicles were found in the region of the gradient expected for the ER (fractions 23-25, Fig 1). The data suggest that the PCTV can deliver their [3H]TG cargo preferentially to the heavy but also the light Golgi fractions but not the ER, which is consistent with our prior findings (15). In summary, these data support our hypothesis that we have isolated a functional intermediate, TG-containing fraction that is post-ER but pre-Golgi and that this fraction delivers TG to the Golgi in a vectorial manner as would be expected of intracellular TG movement in the enterocyte.

                              
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Table 2.   Delivery of [3H]TG from vesicles to ER and Golgi fractions

Apolipoprotein constituents of PCTV, ER, and Golgi. We next wished to determine the apolipoprotein content of the vesicular fraction because it would be likely that a putative PCTV would also contain at least some of the apolipoproteins present in chylomicrons. Apo B48 and apo A-IV were easily identified on Western blotting in the ER, PCTV, Golgi, and chylomicrons (Fig. 3). Compared with apo B48, the PCTV had more apo A-IV than the ER and Golgi and less than the chylomicrons (legend to Fig. 3).


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Fig. 3.   Western blot of apolipoprotein (apo) B48 (top), apo A-I (middle), and apo A-IV (bottom) from various subcellular fractions of intestinal cells. Subcellular fractions were isolated from continuous sucrose gradient (vesicles) or sucrose-step gradient for ER and Golgi (heavy and light Golgi) as described in MATERIALS AND METHODS. Chylomicrons were isolated from lymph of rats infused intraduodenally with triolein. Equal amounts of protein (50 µg), except for chylomicrons which were 5 µg, were electrophoresed by SDS-PAGE, proteins were transblotted to membranes, and appropriate antibody was applied. Apo B48 (top) is identifiable in all fractions. Smaller anti-apo B antibody-positive bands are presumed to be degradation products of apo B48. Anti-apo A-I positive bands (middle) are identified in each fraction, but only a small amount was present in the PCTV. Immunoidentified apo A-IV is shown at bottom. Figure is representative of 3 such studies. Apo B48, apo A-I, and apo A-IV bands were digitized and compared using arbitrary densitometry units: apo A-I/apo B48, ER = 0.77 ± 0.1; heavy Golgi (HG) = 0.68 ± 0.1; light Golgi (LG) = 0.7 ± 0.1; PCTV = 0.09 ± 0.01; chylomicrons (CM) = 1.7 ± 0.2; n = 3; ANOVA P = 0.0002. PCTV had less apo A-I compared with apo B48 than any other fraction (P < 0.02). Apo A-IV/apo B48, ER = 0.68 ± 0.01, HG = 1.04 ± 0.06, LG = 1.15 ± 0.04, PCTV = 1.66 ± 0.11, CM = 5.2 ± 0.13; ANOVA P = 0.0001. Compared with apo B48, PCTV had more apo A-IV than ER and Golgi but less than CM (P < 0.01).

Apo A-I was also easily immunoidentified in the ER, Golgi, and chylomicrons (Fig. 3). Compared with the amount of apo B48 present, nearly the same amount of apo A-I was immunoidentified in the ER and Golgi and twice as much in chylomicrons (legend to Fig. 3). By contrast, little apo A-I was found in the PCTV fraction; the apo A-1/apo B48 ratio in the PCTV was only 12-14% of the ratio in the ER and Golgi and 6% of the ratio in chylomicrons (legend to Fig. 3).

Protein separation by electrophoresis in ER, Golgi, and PCTV. If the PCTV were a unique fraction, then a PCTV-protein pattern that differs from that identified in the ER and Golgi should be found on either SDS-PAGE, 2-D gels, or both. To address this question, equal amounts of protein of PCTV, ER, and Golgi were applied to SDS-PAGE and the results are shown in Fig. 4. Differences between the fractions are obvious. Three protein bands which are unique to the PCTV membrane are identified by arrows.


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Fig. 4.   SDS-PAGE of subcellular fractions from enterocytes. Equal amounts of protein (100 µg) were applied to 4-20% SDS-PAGE from intestinal cell subcellular fractions, which were isolated according to procedures described in MATERIALS AND METHODS. Light Golgi fraction floated at the 0.25/0.86 M sucrose interface and heavy Golgi fraction at the 0.86/1.15 M sucrose of sucrose-step gradient. Differences in protein bands (Coomassie blue stained) between the various fractions are evident. Some proteins unique to PCTV are identified by arrows. Gel is representative of 4 trials.

Because multiple proteins could be present which are represented by a single band on SDS-PAGE, we performed 2-D gels of the ER, light and heavy Golgi, and the PCTV, with the results shown in Fig.5. Again, differences among the ER, Golgi fractions, and the PCTV are clear. Two proteins, unique to the PCTV, are identified by arrows. These fractions presumably contained developing chylomicrons as well as a variety of other proteins in their lumens and therefore the differences noted between fractions could be due to different luminal proteins trafficking through the compartment at the moment of isolation.


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Fig. 5.   Two-dimensional (2-D) gels of various subcellular fractions from enterocytes. 2-D electrophoresis was performed on 10% acrylamide gels of ER (A), heavy Golgi (B), light Golgi (C), and PCTV (D), all of which were isolated as in MATERIALS AND METHODS. Equal amounts of protein were applied to each lane (50 µg). Gels were silver stained. Some proteins unique to PCTV are identified by arrows. Gel is representative of 4 trials.

To test whether membrane proteins were also different between fractions, we incubated each fraction with carbonate (see MATERIALS AND METHODS) to release proteins entrapped within their lumens. The membranes were collected by centrifugation and washed with 0.15 M NaCl to remove the carbonate, and an equal amount of protein from each fraction was separated on 2-D gels. As shown in Fig. 6 many fewer proteins were found in each fraction, indicating that luminal proteins had been released. Again, clear differences between the PCTV and the ER and Golgi membranes were found (two unique proteins in the PCTV fraction and one unique protein in each of the other fractions are identified by arrows), suggesting that different proteins were present in the membranes of each fraction. These data further establish that 1) the vesicle fraction isolated in the less dense region of the continuous sucrose gradient is different from the ER and Golgi; 2) the vesicles contain proteins that are releasable with carbonate, suggesting that some proteins were sequestered intraluminally; and 3) there is a protein-containing fraction that pellets on centrifugation after carbonate treatment of the PCTV, a finding that is consistent with a membrane structure. These data support the vesicular nature of the PCTV and that it is a unique subcellular fraction.


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Fig. 6.   Gels (2-D) of membranes of various subcellular fractions from enterocytes. Subcellular fractions were isolated and treated with carbonate, and ER, Golgi, and PCTV membranes were collected by centrifugation (see MATERIALS AND METHODS). Resulting membranous proteins (50 µg each) were separated by 2-D PAGE (10%), and gels were stained with silver stain. A: ER. B: heavy Golgi. C: light Golgi. D: PCTV. Each subcellular fraction demonstrates unique protein spots. Some of these proteins are indicated by arrows. Figure is representative of 4 such studies.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The present study is an extension of work recently presented from this laboratory which showed that TG and apo B48 could traffic from the ER to the Golgi in a cell-free system (15). The data further suggested that a vesicular mechanism was the likely means by which TG and apo B48 were transported to the Golgi and that both entered the Golgi lumen, supporting the thesis that fusion of the putative vesicle with the Golgi occurred. The delivery of TG to specifically the intestinal Golgi was an ATP, cytosolic protein, and temperature-dependent step. Finally, the physiological importance of the TG and apo B48 transport mechanism was enhanced by our finding that the rate of movement of TG from the ER to the Golgi correlated with the known ability of the intestine to export TG into the lymph as chylomicrons under varying physiological conditions. To further understand this process, the current experiments were directed at identifying the putative TG transport vesicle and partially characterizing it.

The data presented here suggest that we have been able to isolate a vesicle (Fig. 2), which we have named the PCTV. This vesicle is post-ER but pre-Golgi. This thesis is supported by two pieces of evidence: 1) the PCTV is able to deliver TG to the Golgi but not the ER and 2) the vesicle contains only small amounts of apo A-I compared with the ER, Golgi, and chylomicrons. Any post-Golgi vesicle containing prechylomicrons would be expected to contain considerable apo A-I because apo A-I is present both in the Golgi and in chylomicrons (Fig. 3). Indeed we found that the ratio of apo A-I to apo B48 in chylomicrons isolated from the lymph was nearly twice that found in the Golgi (Fig. 3) likely due to nonchylomicron-associated apo B48 which is present in the Golgi. These cell biological data supporting a prechylomicron transport vesicle are consistent with prior electron microscopic observations of Friedman and Cardell (5) who showed that lipid-containing vesicles were present between the ER and the Golgi in lipid-absorbing rats.

In the present and our prior work (15), we have begun to characterize the movement of TG and apolipoproteins between the ER and the Golgi in a cell-free system in the expectation that we will be able to overcome some of the obstacles that have slowed detailed investigation of the intracellular movement of chylomicron-TG and its regulation.

To further our studies the isolation of the putative transport vesicle was required. This proved to be difficult because homogenization using Potter Elveheim pestles, cellular disruption using a Parr bomb, or sonication did not produce a homogenate from which we were able to separate a putative vesicle from the ER and/or Golgi by density gradients. To retain the ER and Golgi in situ while releasing the vesicles, we developed a method in which the apical portion of the enterocytes was avulsed and the mucosa stirred in buffer. This enabled us to obtain a purified preparation of vesicles which contained TG at electron microscopy, apo B48 by Western blotting, and delivered its TG vectorially to the Golgi thus satisfying our minimal criteria for successful isolation of the PCTV.

Once the vesicles were isolated, we sought to separate the proteins of the vesicle to determine if they differed from ER and Golgi proteins, thus further differentiating the PCTV from the ER and Golgi. This was done first by SDS-PAGE and 2-D gels of both whole organelles and vesicles and subsequently by 2-D gels of their membranes with their luminal contents removed. As shown in Figs. 4-6, clear differences were observed between the PCTV and the ER and Golgi in each case. These differences in protein composition between the PCTV and the ER and Golgi support our thesis that we have isolated a unique fraction. Our vesicle can also be differentiated from the mitochondria-associated membrane fraction described by Rusinol et al. (32) by marker enzyme analyses and differences in sedimentation characteristics.

It would appear from the size, mostly 200 nm, and shape of the morphologically apparent TG component of the PCTV that there is only one chylomicron per vesicle. This would suggest that the movement of the vesicles from the ER to the Golgi is rapid because a large amount of TG needs to be transported quickly. The short distance to be traveled between the two organelles as suggested by Friedman and Cardell (5) and the speed at which vesicles move from the ER to the Golgi (39), suggest that adequate movement of TG to the Golgi could occur by the proposed vesicular mechanism.

Of particular interest is the relative lack of apo A-I in the PCTV compared with the ER and Golgi. Its presence in both ER (where it is synthesized) and Golgi (where it is associated with chylomicrons) was expected. However, its presence in only small amounts in the PCTV was unforeseen. We expected apo A-I to be present in the PCTV because apo A-I is water soluble, suggesting that it would have access to the surface of the developing chylomicron in the lumen of the ER, and once exposed to the surface of the chylomicron, it would be expected to bind because it has been shown that apo A-I binds spontaneously to suitable lipid surfaces (30). Because apo A-I is found in the PCTV in such small amounts compared with the ER, we suggest that adequate binding of apo A-I to the chylomicron surface does not occur in the ER lumen, otherwise apo A-I would be present in the same ratio to apo B48 as it is in the ER. In sum, these data imply that apo A-I utilizes a mechanism to get from the ER to the Golgi other than the PCTV. The fact that apo A-I traverses the intracellular space from ER to Golgi separate from the PCTV provides a mechanism for understanding why cells maintain their ability to excrete apo A-I even when apo B secretion is suppressed (6, 9, 11).

The only other investigation of the movement of lipids between the ER and the Golgi was performed in liver fractions by Moreau et al. (21) in which phospholipids and cholesterol, but not TG, were shown to move in an ATP-dependent step from the ER to the Golgi which had been immobilized on neoprene strips. The fact that the intestine transports TG to the Golgi via a different mechanism from the liver is another difference between the liver and intestine with respect to lipid and lipoprotein biosynthesis. For example the liver does not express intestinal fatty acid binding protein (34), the mature liver does not express MG acyltransferase activity (4), and the liver obtains TG for its TG-rich lipoprotein, very low-density lipoprotein, differently than does the intestine for chylomicrons. In the liver, TG from the TG storage pool is first partially hydrolyzed to diacylglycerol and subsequently resynthesized to TG at the level of the ER (20). By contrast, the intestine recruits TG for chylomicron formation from newly synthesized TG, preferentially using dietary MG as the glyceride-glycerol precursor. This TG, even if it is blocked from passage to the Golgi by the nonionic detergent Pluronic L-81, is only minimally hydrolyzed before its export into the lymph as chylomicron-TG (10).

The vesicular membrane of the PCTV, which surrounds the developing chylomicron, may serve to protect the chylomicron-TG from the action of the intestinal alkaline-active lipase. We have described and isolated (28, 29) this enzyme and found that it is present in the cytosolic compartment of predominantly the villous tips of the proximal intestine, the major site of lipid absorption. In unreported studies, we have cloned and sequenced this lipase (R. H. Rao, J. Mahan, and C. M. Mansbach, unpublished observations) and find that its sequence matches perfectly with rat pancreatic lipase. Pancreatic lipase is unlikely to penetrate and hydrolyze membranous structures because it is unable to hydrolyze phospholipid monolayers at pressures above 20 dynes/cm, well below the lateral surface pressure of membranes (37). Thus it is likely that one function of the PCTV is to protect its enclosed chylomicron-TG from hydrolysis as it transits the cytosol from the ER to the Golgi.

In summary, we have isolated a vesicular intermediate from the intestinal mucosa that can transfer TG to intestinal Golgi in a unidirectional manner. Four pieces of evidence lead us to conclude that we have identified a unique subcellular fraction, different from the ER, Golgi, and lysosomes: 1) the marker enzyme studies differentiate the PCTV from ER, Golgi, and lysosomes; 2) the sedimentation characteristics suggest a TG-filled vesicle because of their light buoyant density compared with ER and Golgi fractions; 3) one- and two-dimensional PAGE analyses demonstrate different protein patterns in the PCTV vs. the ER and Golgi; and 4) the relative lack of apo A-I to apo B48 in the PCTV compared with the ER and Golgi fractions. Understanding the role of the proteins that are specific to the surface of the PCTV may provide insight into how the intestine controls chylomicron output rates.


    ACKNOWLEDGEMENTS

This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38760 and in part by the Veterans Affairs Medical Research Funds.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. M. Mansbach II, Univ. of Tennessee, 951 Court Ave., Rm. 555 Dobbs, Memphis, TN 38163.

Received 2 June 1998; accepted in final form 20 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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