6
THE JOURNAL OF B~OLOOICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry end Molecular Biology, Inc. Vol. 265, No. 4, Issue of February 5, pp. 1919-1923,199O Printed in U. 5’. A. Cholesterol and Vesicular Stomatitis Virus G Protein Take Separate Routes from the Endoplasmic Reticulum to the Plasma Membrane* (Received for publication, September 5, 1989) Lenore Urbani and Robert D. Simoni From the Department of Biological Sciences, Stanford Uniuersity, Stanford, California 94305-5020 Transport of newly synthesized cholesterol and ve- sicular stomatitis virus G protein from the endoplasmic reticulum to the plasma membrane is interrupted by incubation at 15 ‘C. Under this condition the newly synthesized molecules accumulate in both the endo- plasmic reticulum (ER) and a subcellular vesicle frac- tion of low density called the lipid-rich vesicle fraction. The material in the lipid-rich vesicle fraction appears to be a post-ER intermediate in the transport process to the plasma membrane (PM). Although both newly synthesized cholesterol and G protein accumulate in this intermediate compartment at 15 “C, suggesting co- transport, treatment with Brefeldin A does not affect cholesterol transport to the PM, whereas it strongly inhibits G protein transport. We conclude that choles- terol and G protein leave the ER in separate vesicles, the cholesterol containing vesicles bypass the Golgi apparatus and proceed to the PM, whereas G protein containing vesicles follow the well documented Golgi route to the cell surface. Cholesterol synthesis is a complex biosynthetic process requiring the participation of more than 27 enzymes, many of which are located in the endoplasmic reticulum. Although the exclusivity of this localization has been questioned in tissue culture cells (l), others have demonstrated that all membrane bound cholesterol biosynthetic enzymes in liver are localized in the endoplasmic reticulum (2). After synthesis, cholesterol must be transported from the endoplasmic reticulum to the plasma membrane, where 90% of cellular cholesterol is found in cultured human fibroblasts (3). Over the last several years we and others (4-6) have devel- oped assays which measure the transport of cholesterol from the endoplasmic reticulum to the plasma membrane. Our experiments have demonstrated that the tXh for cholesterol transport from the endoplasmic reticulum to the plasma mem- brane is about 10 min (4,5). The process is energy-dependent, and energy depletion results in accumulation of newly syn- thesized cholesterol in the endoplasmic reticulum (4,5). Cho- lesterol transport is also interrupted by incubation of cells at 15 “C, resulting in accumulation of newly synthesized choles- terol in the endoplasmic reticulum and in a subcellular frac- tion of vesicles of low density (5). We have proposed that this lipid-rich vesicle fraction, the LRVF,’ contains an interme- * This work was supported by American Cancer Society Grant CD 252 and National Institutes of Health Grant HL26502. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: LRVF, lipid-rich vesicle fraction; BFA, Brefeldin A; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; Hepes, 4-(2-hydroiyethyl)-1-piperazineethanesulfonic acid; VSV, vesicular stomatitis virus: SDS, sodium dodecvl sulfate; Endo- H, endo+N-acetylglucosaminidase H; PM, plasma-membrane. diate in the intracellular transport of cholesterol from the site of synthesis in the endoplasmic reticulum to the final desti- nation in the plasma membrane (5). In this report we show that G protein of vesicular stomatitis virus, a model plasma membrane protein, also accumulates in the LRVF during incubation at 15 “C. The fact that both newly synthesized cholesterol and G protein accumulate in the LRVF during incubation at 15 “C raised the possibility that these two classes of molecules that share a subcellular origin and desti- nation also share a step in the transport process. In an attempt to resolve this possibility we have used the lipophilic fungal metabolite Brefeldin A (BFA), which allowed us to distinguish transport of cholesterol from that of plasma membrane pro- tein transport. Brefeldin has increasingly been the focus of protein transport studies. It was shown to disrupt intracellular protein transport and appears to cause the accumulation of proteins in the ER and disassembly of the Golgi apparatus (7, 8). The results reported in this paper demonstrate that the transport of G protein to the plasma membrane is blocked by Brefeldin treatment, but cholesterol transport is unaffected. EXPERIMENTAL PROCEDURES MateriaIs-[‘HIacetate (3.4 Ci/mmol), [3H]UDP-galactose (10.2 Ci/mmol). 11.2-‘4Clacetate (50 mCi/mmol). r&-3-hvdroxv-3- ~ethyl[~-‘4C]g~&&l coe;lzyme A’ (26.2 n&i/m,ol) L-[35i]methio- nine (1151 Ci/mmol) were obtained from Du Pont-New England Nuclear. Tissue culture media and fetal calf serum were from Gibco Laboratories. Brefeldin A was a eift from Dr. Richard Klausner. Silica Gel-G TLC plates, 250-pm were from Analtech, Inc. (Newark, DE). All other reagents were highest purity available from common vendors. Cell Culture-Chinese hamster ovary (CHO) cells were grown in monolayer in a-minimal Eagle’s medium supplemented with 10% lipoprotein-depleted fetal calf serum (4, 5) as described previously. Labeling of Cellular Cholesterol-Twenty-two hours before the ex- periment, confluent 150-mm plates of cells were trypsinized, split 1:2, and grown in pyruvate-free medium supplemented with 10% lipid- depleted serum (4), with 1 &i of [14C]acetate/plate. To prepare cells for radiolabeling, cells were again trypsinized and resuspended in pyruvate-free medium, buffered with 15 mM Hepes buffer, pH 7.0, at a concentration of 10’ cells/ml. Cells were preincubated at 15 “C for 10 min; the pulse labeling was initiated by addition of 50 &i/ml of [3H]acetate and the chase by addition of 2.0 M acetate to a final concentration of 20 mM. The times of the pulse and chase are specified in each experiment. Infection with Vesicular Stoma&is Virus and Labeling of G Pro- tein-cells were infected with vesicular stomatitis virus (VSV) by the method of Fries et al. (9). 135 min after infection, cells were trypsinized, pulse-labeled at 15 “C with [35S]methionine (30.0 &i/ ml), and chased with the addition of methionine to a final concentra- tion of 2.5 mM. G protein was quantified by densitometric scanning of autoradiographic images of the proteins separated by SDS-poly- acrylamide gel electrophoresis analysis. Samples treated with Endo- H were boiled for 3 min with equal volumes of 0.1 M Tris-HCl, pH 6.8, 2% SDS, and 30 mM dithiothreitol. Three milliunits of End&H dissolved in 0.3 M sodium citrate. DH 5.5. 0.1% SDS were added to each cooled sample and incubated &ernight at 37 ‘C. Control samples 1919 by guest on April 20, 2018 http://www.jbc.org/ Downloaded from

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THE JOURNAL OF B~OLOOICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry end Molecular Biology, Inc.

Vol. 265, No. 4, Issue of February 5, pp. 1919-1923,199O Printed in U. 5’. A.

Cholesterol and Vesicular Stomatitis Virus G Protein Take Separate Routes from the Endoplasmic Reticulum to the Plasma Membrane*

(Received for publication, September 5, 1989)

Lenore Urbani and Robert D. Simoni From the Department of Biological Sciences, Stanford Uniuersity, Stanford, California 94305-5020

Transport of newly synthesized cholesterol and ve- sicular stomatitis virus G protein from the endoplasmic reticulum to the plasma membrane is interrupted by incubation at 15 ‘C. Under this condition the newly synthesized molecules accumulate in both the endo- plasmic reticulum (ER) and a subcellular vesicle frac- tion of low density called the lipid-rich vesicle fraction. The material in the lipid-rich vesicle fraction appears to be a post-ER intermediate in the transport process to the plasma membrane (PM). Although both newly synthesized cholesterol and G protein accumulate in this intermediate compartment at 15 “C, suggesting co- transport, treatment with Brefeldin A does not affect cholesterol transport to the PM, whereas it strongly inhibits G protein transport. We conclude that choles- terol and G protein leave the ER in separate vesicles, the cholesterol containing vesicles bypass the Golgi apparatus and proceed to the PM, whereas G protein containing vesicles follow the well documented Golgi route to the cell surface.

Cholesterol synthesis is a complex biosynthetic process requiring the participation of more than 27 enzymes, many of which are located in the endoplasmic reticulum. Although the exclusivity of this localization has been questioned in tissue culture cells (l), others have demonstrated that all membrane bound cholesterol biosynthetic enzymes in liver are localized in the endoplasmic reticulum (2). After synthesis, cholesterol must be transported from the endoplasmic reticulum to the plasma membrane, where 90% of cellular cholesterol is found in cultured human fibroblasts (3).

Over the last several years we and others (4-6) have devel- oped assays which measure the transport of cholesterol from the endoplasmic reticulum to the plasma membrane. Our experiments have demonstrated that the tXh for cholesterol transport from the endoplasmic reticulum to the plasma mem- brane is about 10 min (4,5). The process is energy-dependent, and energy depletion results in accumulation of newly syn- thesized cholesterol in the endoplasmic reticulum (4,5). Cho- lesterol transport is also interrupted by incubation of cells at 15 “C, resulting in accumulation of newly synthesized choles- terol in the endoplasmic reticulum and in a subcellular frac- tion of vesicles of low density (5). We have proposed that this lipid-rich vesicle fraction, the LRVF,’ contains an interme-

* This work was supported by American Cancer Society Grant CD 252 and National Institutes of Health Grant HL26502. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: LRVF, lipid-rich vesicle fraction; BFA, Brefeldin A; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; Hepes, 4-(2-hydroiyethyl)-1-piperazineethanesulfonic acid; VSV, vesicular stomatitis virus: SDS, sodium dodecvl sulfate; Endo- H, endo+N-acetylglucosaminidase H; PM, plasma-membrane.

diate in the intracellular transport of cholesterol from the site of synthesis in the endoplasmic reticulum to the final desti- nation in the plasma membrane (5). In this report we show that G protein of vesicular stomatitis virus, a model plasma membrane protein, also accumulates in the LRVF during incubation at 15 “C. The fact that both newly synthesized cholesterol and G protein accumulate in the LRVF during incubation at 15 “C raised the possibility that these two classes of molecules that share a subcellular origin and desti- nation also share a step in the transport process. In an attempt to resolve this possibility we have used the lipophilic fungal metabolite Brefeldin A (BFA), which allowed us to distinguish transport of cholesterol from that of plasma membrane pro- tein transport. Brefeldin has increasingly been the focus of protein transport studies. It was shown to disrupt intracellular protein transport and appears to cause the accumulation of proteins in the ER and disassembly of the Golgi apparatus (7, 8). The results reported in this paper demonstrate that the transport of G protein to the plasma membrane is blocked by Brefeldin treatment, but cholesterol transport is unaffected.

EXPERIMENTAL PROCEDURES MateriaIs-[‘HIacetate (3.4 Ci/mmol), [3H]UDP-galactose

(10.2 Ci/mmol). 11.2-‘4Clacetate (50 mCi/mmol). r&-3-hvdroxv-3- ~ethyl[~-‘4C]g~&&l coe;lzyme A’ (26.2 n&i/m,ol) L-[35i]methio- nine (1151 Ci/mmol) were obtained from Du Pont-New England Nuclear. Tissue culture media and fetal calf serum were from Gibco Laboratories. Brefeldin A was a eift from Dr. Richard Klausner. Silica Gel-G TLC plates, 250-pm were from Analtech, Inc. (Newark, DE). All other reagents were highest purity available from common vendors.

Cell Culture-Chinese hamster ovary (CHO) cells were grown in monolayer in a-minimal Eagle’s medium supplemented with 10% lipoprotein-depleted fetal calf serum (4, 5) as described previously.

Labeling of Cellular Cholesterol-Twenty-two hours before the ex- periment, confluent 150-mm plates of cells were trypsinized, split 1:2, and grown in pyruvate-free medium supplemented with 10% lipid- depleted serum (4), with 1 &i of [14C]acetate/plate. To prepare cells for radiolabeling, cells were again trypsinized and resuspended in pyruvate-free medium, buffered with 15 mM Hepes buffer, pH 7.0, at a concentration of 10’ cells/ml. Cells were preincubated at 15 “C for 10 min; the pulse labeling was initiated by addition of 50 &i/ml of [3H]acetate and the chase by addition of 2.0 M acetate to a final

concentration of 20 mM. The times of the pulse and chase are specified in each experiment.

Infection with Vesicular Stoma&is Virus and Labeling of G Pro- tein-cells were infected with vesicular stomatitis virus (VSV) by the method of Fries et al. (9). 135 min after infection, cells were trypsinized, pulse-labeled at 15 “C with [35S]methionine (30.0 &i/ ml), and chased with the addition of methionine to a final concentra- tion of 2.5 mM. G protein was quantified by densitometric scanning of autoradiographic images of the proteins separated by SDS-poly- acrylamide gel electrophoresis analysis. Samples treated with Endo- H were boiled for 3 min with equal volumes of 0.1 M Tris-HCl, pH 6.8, 2% SDS, and 30 mM dithiothreitol. Three milliunits of End&H dissolved in 0.3 M sodium citrate. DH 5.5. 0.1% SDS were added to each cooled sample and incubated &ernight at 37 ‘C. Control samples

1919

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1920 Cholesterol and VSV G Protein

without Endo-H digestion were incubated overnight with the sodium citrate, SDS buffer.

Cell Fractionation-Following the chase, the cells were mixed with 2 volumes of ice-cold phosphate-buffered saline, centrifuged for 3 min at 4 “C at 600 X g, and washed once with 50 ml of cold phosphate- buffered saline. The pellet was resuspended in 4 ml of 10 mM Tris- HCI buffer, pH 7.4, and the cells isolated by centrifugation for 5 min at 1900 X g. The cells were gently resuspended in 2 ml of the same buffer, transferred to a 7-ml glass Dounce homogenizer and incubated on ice for 5 min. Leupeptin, dithiothreitol, and phenylmethylsulfonyl fluoride were added to a final concentration of 0.1, 1.0, and 0.1 mM, respectively. After one stroke of a tight-fitting pestle, 2 ml of 0.3 M sucrose in 10 mM Tris-HCl buffer, pH 7.4, was quickly added. The cells were further disrupted with 19 more strokes in the homogenizer. 2 ml of homogenate was mixed with 6.5 ml of 63.5% w/w sucrose in 1 mM Tris-EDTA (TE) buffer, pH 8.0. Leupeptin, dithiothreitol, and phenylmethylsulfonyl fluoride were added to previously stated con- centrations. The homogenate was overlayed with 7.5-ml steps of 38, 31, and 23% sucrose w/w and 1 mM TE buffer, pH 8.0. Gradients were centrifuged for 3 h at 4 “C in an SW 28 rotor at 24,000 rpm. Fractions were collected from the bottom of the tube using a poly- staltic pump. Fractions were then mixed with 3 volumes of 1 mM TE buffer, pH 8.0, pelleted for 1 h at 40,000 rpm in a 60 Ti rotor, and resuspended in 0.5 ml of the same buffer.

Lipid Analysis-200 ~1 of the sucrose gradient fractions and 5 ~1 of whole cell homogenate were extracted overnight in 3.0 ml of chloro- form/methanol 1:l. An equal volume of 0.04 N HCl was then added, and the sample was vortexed and centrifuged at 300 x g for 7 min. The chloroform phase was dried with nitrogen, brought to 80 ~1 with chloroform/methanol 1:l and applied to a Silica Gel-G TLC plate. Plates were developed in hexane, ether, acetic acid, 84:20:2 and visualized with iodine vapor. Cholesterol spots were scraped and counted in a liquid scintillation counter. The purity of the cholesterol synthesized in the manner described in this paper, and isolated by TLC, was verified previously by high performance liquid chromatog- raphy analysis (4).

Data Analysis-The results of cell pulse-chase fractionation exper- iments are expressed as relative [3H]/[‘4C]cholesterol calculated as [3H]/pC]cholesterol in the membrane fractions of the sucrose gra- dients, divided by the [3H]/[‘4C]cholestero1 of the crude homogenate applied to the sucrose gradient. The use of such relative values is necessary because the chase of radioactive acetate is not immediately effective and the values must be corrected for the continuing synthesis that occurs during the chase. We have described this previously (4, 5).

Brejeldin A Incubation-Brefeldin A was dissolved in 100% meth- anol to a stock concentration of 5 mg/ml and stored at -20 “C. It was added to CHO cells 30 min before the pulse. The medium containing the Brefeldin was removed and replaced with fresh medium contain- ing Brefeldin, for the pulse and again for the chase. This was neces- sary because Brefeldin has been shown to be degraded (7).

DEAE Bead Isolation of Plasma Membranes-The DEAE bead method for the isolation of plasma membranes was used to measure protein and cholesterol transport according to the procedure of DeGrella and Simoni (4).

RESULTS

Subcellular Fractionation of CHO Cells in the Assay of Cholesterol Transport-CHO cells grown in lipid free medium provide an experimental system of cells that are dependent upon de nouo biosynthesis and intracellular transport of cho- lesterol for growth. One assay for cholesterol transport used in this report and discussed below involves a pulse-chase labeling of newly synthesized cholesterol with [3H]acetate and subcellular fractionation to determine the intracellular local- ization of newly synthesized cholesterol. Using cholesterol and activities of hydroxymethylglutaryl-CoA reductase and galactosyl transferase as markers for plasma membrane (PM), endoplasmic reticulum (ER), and the Golgi apparatus, respec- tively, it can be seen in Fig. 1 that the fractions enriched in these subcellular markers are separated.

Znhibition of Cholesterol Transport as 15 “C-Extended pre- labeling of cells with [“QZ]acetate results in the synthesis and distribution of [YJ]cholesterol of constant specific activity

2 4 6 6 IO 12 14 FRACTION

FIG. 1. Fractionation of CHO cell homogenates on sucrose gradients. Gradients were run and assays performed as described under “Experimental Procedures.” Hydroxymethylglutaryl (HMG)- CoA reductase activity is given as nmoles mevalonate formed/min/ mg and galactosyltransferase is given as 3H counts/min/pg protein.

and serves as a measure of cholesterol mass, whereas pulse- chase labeling of cells with [3H]acetate distinguishes newly synthesized [3H]cholesterol. Thus, 3H/14C ratio is a relative measure of enrichment of newly synthesized [3H]cholesterol in any subcellular fraction. When cells are incubated with [3H]acetate for 30 min or more (about three times the tlh of transport), at 37 “C, the newly synthesized [3H]cholesterol reaches a subcellular distribution identical to that of the [‘“Cl cholesterol, indicating that intracellular transport is complete (data not shown). If, however, cells are incubated with [3H] acetate at 15 “C, newly synthesized cholesterol does not arrive at the PM but instead accumulates primarily in the ER fraction and in a low density fraction that we have called the LRVF as indicated by a high [3H]/[‘4C]cholestero1 ratio shown in Fig. 2. In contrast, the plasma membrane and Golgi fractions are deficient in newly synthesized cholesterol and share a low [3H]/[14C]cholestero1 ratio. If the synthesis at 15 “C is followed by a 37 “C chase, the [3H]cholesterol accu- mulated at 15 “C! in both the ER and the LRVF proceeds to the plasma membrane attaining the same distribution as [‘“Cl cholesterol (Fig. 2). Furthermore, kinetic data indicate that the rate of appearance and disappearance of newly synthe- sized cholesterol in the LRVF is consistent with the overall transport rate, suggesting that the LRVF could be an inter- mediate in the transport process (10).

G Protein of Vesicular Stomatitis Virus Ako Accumulates in the LRVF during Synthesis at 15 “C-As discussed above, we have shown that incubation of CHO cells at 15 “C interrupts cholesterol transport to the plasma membrane and results in accumulation of newly synthesized cholesterol not only in the ER, but in the LRVF as well. We feel this LRVF material

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Cholesterol and VSV G Protein

y 14. 9 ,2 ‘O-

Y 6-

5i d .2-

[L 2 4 6 8 IO I2 14 FRACTIONS

FIG. 2. Subcellular localization of newly synthesized cho- lesterol during synthesis at 15 “C. Cells were pulsed labeled with [“HIacetate for 60 min at 15 “C. One aliquot was then chased for 60 min at 15 “C (0) while the other was chased for 90 min at 37 “C (W). Homogenates were prepared and fractionated on sucrose gradients as described under “Exnerimental Procedures.” The abbreviations in this and all subsequent sucrose gradient profiles are: ER, endoplasmic reticulum; PM, plasma membrane; LRVF, lipid-rich vesicle fraction; GOL, Golgi and represent the peak fractions of each marker.

FRACTlOhl

FIG. 3. Accumulation of G protein during incubation at 15 “C. VSV-infected cells were pulse-labeled with [%]methionine for 90 min at 15 “C followed by a chase for 60 min at 15 “C (0) or 37 “C (m). Cell homogenates were prepared and fractionated as de- scribed under “Experimental Procedures.” The amount of G protein present in various fractions was determined as described under “Ex- perimental Procedures.”

may represent an intermediate in the cholesterol transport process. Saraste and Kuismanen (11) demonstrated at 15 “C in Semliki forest virus-infected baby hamster kidney cells an intermediate compartment for protein transport between the endoplasmic reticulum and the Golgi. They obtained immu- nocytochemical data demonstrating viral proteins in lOO-nm vesicles that appeared to be budding from the ER and postu- lated that these vesicles could be the pre-Golgi transport intermediate. They also found the viral glycoprotein to be Endo-H-sensitive when synthesized at 15 “C, indicating the protein had not been processed by enzymes of the medial Golgi (12). Beckers and Balch (13) have demonstrated a post- ER, pre-cis Golgi compartment accumulating in CHO 15B cells after incubation at 15 “C. These observations that both cholesterol and protein transport are interrupted at 15 “C suggest that cholesterol transport and protein transport might share a common pathway. In order to test this hypothesis, we measured the subcellular distribution of newly synthesized G protein in cells incubated at 15 “C, to determine whether its distribution was similar to that of cholesterol. The results, presented in Fig. 3, show that after synthesis at 15 “C, G protein has a subcellular distribution similar to that of newly synthesized cholesterol, indicating an enrichment of G protein in the ER and the formation of a G protein containing vesicle

fraction that co-fractionates with the LRVF. G protein in all fractions was found to be Endo-H-sensitive, indicating a pre- Golgi compartment (data not shown). If synthesis of G protein at 15 “C is followed by a 60-min chase at 37”C, G protein is lost from the ER and LRVF and is found primarily in the Golgi/plasma membrane fractions (see Fig. 3) and becomes Endo-H-resistant (data not shown).

The presence of both cholesterol and G protein in the same vesicle would imply that a common route from the ER through the Golgi is taken on the way to the PM. It is interesting to speculate that integral plasma membrane proteins might pro- vide the signal for the cholesterol-rich vesicles to proceed to the Golgi and on to the plasma membrane. Since newly synthesized cholesterol (Fig. 2) and G protein (Fig. 3) cofrac- tionate in the LRVF compartment (after synthesis at 15 “C) and possibly share an intracellular pathway, it became critical to determine if they exist in the same vesicles or in separate vesicles of similar density as they exit from the ER. Exhaus- tive attempts to resolve this question by antibody purification of these vesicles were, however, unsuccessful.

The Effects of Brefeldin A on G Protein and Cholesterol Synthesis and Transport-Since our attempts to physically separate cholesterol and G protein containing vesicles from the LRVF fractions were unsuccessful, we have functionally separated the two processes using BFA. The rapid disassembly of the cis and medial Golgi cisternae by BFA disrupts protein transport and proteins accumulate in the ER (7,8).

In order to document the effect of BFA on protein transport in our experimental system, VSV-infected cells were pulse- labeled at 37 “C in the presence of several concentrations of Brefeldin, from 0.01 to 5.0 rg/ml. A concentration as low as 0.1 pg/ml was sufficient to render the G protein Endo-H- sensitive, indicating that transport to the Golgi apparatus had not occurred (data not shown). We also observed a 67% reduction in the rate of G protein synthesis with 1.0 pg/ml of BFA, a finding which differs from that of Misumi et al. (14), where up to 1 pg/ml of Brefeldin had little effect on protein synthesis in cultured rat hepatocytes. Glycoprotein sensitivity to Endo-H in BFA-treated cells is in agreement with other investigators (14, 15) but differs from the data by Doms et al. (8).

The effect of Brefeldin on G protein transport was deter- mined by isolation of plasma membranes from VSV-infected cells, using the DEAE bead method as described previously

12 3 45 67

M(-m - -) )r)M 15” pulse

37”Chase Endo-H

BFA

FIG. 4. Brefeldin A inhibits G protein transport. Cells were pulse-labeled with [““Slmethionine for 60 min at 15 “C and subse- quently chased for 60 min at 15 “C or 60 min at 37 “C as indicated in the figure. Brefeldin when present was used at 1 pg/ml. The data presented in lanes l-3 were identical whether or not Brefeldin was present. Cells (lanes I and 2), plasma membranes (lanes 34, or virus isolated from medium (lanes 6 and 7) were analyzed for G protein and Endo-H sensitivity as described under “Experimental Proce- dures.” G, N, NS, and M are four of five viral proteins synthesized by VSV infected CHO cells.

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1922 Cholesterol and VS V G Protein

(4, 5), and the results are presented in Fig. 4. VSV-infected cells were pulse labeled with [35S]methionine for 1 h at 15 “C. When the G protein content of the cells is examined, it is observed that all the G protein is Endo-H-sensitive, as one would expect from a 15 “C incubation (see lanes 1 and 2 in Fig. 4). If plasma membranes are isolated from these cells, there is no detectable G protein present (see lane 3 Fig. 4). The presence of Brefeldin does not alter these results. If, however, the 15 “C pulse is followed by a 37 “C chase and the plasma membranes isolated, significant amounts of G protein are found in the plasma membrane compared with Brefeldin- treated cells (see lane 5 Fig. 4). When Brefeldin is present at 1 pg/ml, the amount of G protein in the plasma membrane after incubation at 37 “C is reduced to less than 20% of the control (see lane 4 Fig. 4). These results demonstrate that 1 pg/ml of Brefeldin A is sufficient to virtually abolish G protein transport to the plasma membrane. This is further supported by examination of the G protein content of virus that is found in the medium. As shown in Fig. 4, lane 6, virions produced in the absence of BFA contain G protein, whereas those produced in the presence of BFA are devoid of G protein (Fig. 4, lane 7) as demonstrated previously by Takasuki and Ta- mura (15).

In contrast to the inhibition of G protein synthesis and transport, BFA was found to have no effect on cholesterol synthesis or transport. The cells were pulse-labeled with [3H] acetate at 15 “C for 1 h at 0, 0.5, 2.0, and 5.0 pg/ml of BFA. After a l-h chase at 37 “C! with or without BFA, subcellular fractionation indicated identical cholesterol synthesis at all Brefeldin concentrations and the bulk of the newly synthe- sized cholesterol was present in the PM fractions (Fig. 5).

To determine if BFA affected the rate of cholesterol trans- port, we used the DEAE bead method for isolation of plasma membranes as described previously (4, 5). Cells were pulse- labeled for 1 h at 15 “C with [3H]acetate to allow accumulation of newly synthesized cholesterol in the ER and the LRVF and then chased with or without 1 pg/ml Brefeldin at 37 “C. As seen in Fig. 6, Brefeldin had no effect on the rate of cholesterol transport.

DISCUSSION

The evidence reported in this paper suggests that with incubation at 15 “C! we have isolated a pre-Golgi transport intermediate that is a heterogeneous mixture of lipid-rich vesicles containing, in separate vesicles, newly synthesized

23 00 d k 60 LT y 40

2 0 20

,’ 2 4 6 8 IO 12 14

FRACTIONS FIG. 5. Brefeldin treatment does not affect subcellular cho-

lesterol distribution. Control CHO cells received 15 pl of methanol, and an equal number of cells were incubated for 30 min at 37 “C with 15 ~1 of Brefeldin to a final concentration of 5.0 pg/ml. Cells were pulse-labeled with f3H]acetate for 60 min at 15 “C in the presence grid absence of Brefeldin. The cells were pelleted, resuspended in fresh medium with (0) and without Brefeldin (0). and chased for 60 min at 37 “C. Homogenates were prepared and fractionated on sucrose gradients as described under “Experimental Procedures.”

FIG. 6. Brefeldin does not affect cholesterol transport. Cells were preincubated with 1 rg/ml of Brefeldin and pulse-labeled as described previously in Fig. 5. Aliquots of Brefeldin-treated (U) and -untreated (0) cells were removed at the indicated times of chase at 37 “C. Plasma membrane was isolated with DEAE beads (4) and de nouo cholesterol synthesis measured as described under “Experimen- tal Procedures.”

RER SER PM

GOLGI ’

FIG. 7. G protein and cholesterol (CHOL) are found in sep- arate vesicles. G urotein vesicles may bud off from the rough ER (RER), move through the Golgi, and be Horted to the PM. Cholekerol- rich vesicles may be derived from smooth ER (SER) and proceed directly to the PM.

cholesterol or G protein in the high mannose form. This vesicle fraction, the LRVF, does not share organelle enzyme markers of the Golgi or the ER, but its accumulation at 15 “C and the Endo-H sensitivity of G protein suggests it to be a compartment between the ER and the Golgi. This low density vesicular material may be similar to that described previously by Lodish et al. (16) in a study of protein transport in HepG2 cells (16).

Brefeldin A provides a unique tool to distinguish protein and cholesterol transport. Although both cholesterol and G protein accumulate in the LRVF at 15 “C, newly synthesized cholesterol in BFA-treated cells is transported to the PM at a normal rate, whereas G protein transport is severely inhib- ited. Brefeldin has been shown to cause disassembly of the cis and medial Golgi cisternae, and recycling of cis and medial enzymes back to the ER (8). Because newly synthesized cholesterol was found at the PM in the presence of BFA, the Golgi must not be an obligate intermediate in the sorting/ trafficking of cholesterol to the PM. Monensin, an ionophore that disrupts the trans Golgi elements and the transport of some proteins, was shown previously to have no effect on cholesterol transport (5), consistent with our view that the Golgi is not required for cholesterol transport.

The implication of these results are presented diagramati- tally in Fig. 7. Since our data demonstrate that cholesterol and G protein are in separate vesicles in the earliest detectable

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post-ER compartment, the separation of the two ER-derived 5. Kaplan, M. R., and Simoni, R. D. (1985) J. Cell Biol. 101, 446-

biosynthetic products must occur within the ER. This could 453

be the result of spatial separation of the biosynthetic reactions 6. Lange, Y., and Matthies, H. J. G. (1984) J. Biol. Chem. 259,

or some other mechanism of sequestering the molecules prior 14624-14630

to export. The former seems likely, since it has long been 7. Fujiwara, T., Kimimitsu, O., Yokota, S., Takatsuki, A., and

Ikehara, Y. (1988) J. Biol. Cbem. 263, 1854518552 known that lipid biosynthetic enzymes are generally enriched 8. Doms, R. W., Russ, G., and Yewdell, J. W. (1989) J. Cell Biol. in smooth ER membranes. 109,61-72

9. Fries, E., and Rothman, J. E. (1980) Proc. Natl. Acad. Sci. U. S. Acknowbrdgments-We thank Richard G. W. Anderson for sug- A. 77,3870-3874

gesting the use of Brefeldin and Richard Klausner for a generous 10. Simoni, R. D. (1988) Biological Membranes: Aberrations in Mem-

supply of Brefeldin. We also thank Shoshana Bar-Nun and Michael brane Structure and Function, pp. 29-41, Alan R. Liss, New

Lewis for critically reviewing this manuscript. York 11. Saraste. J.. and Kuismanen. E. (1984) Cell 38. 535-549

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