Assembly of the Endoplasmic Reticulum Phospholipid Bilayer

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 34, Issue of December 5, pp. 16495-16502,1967 Printed in U. S. A.

Assembly of the Endoplasmic Reticulum Phospholipid Bilayer TRANSPORTERS FOR PHOSPHATIDYLCHOLINE AND METABOLITES*

(Received for publication, June 12, 1987)

Yoichi Kawashimal and Robert M. Be118 From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Phosphatidylcholine is synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported to the lumenal monolayer by a protein transporter, a phosphatidylcholine “flippase” (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). Since the endoplasmic reticulum contains enzymes involved in phosphatidylcholine turnover that have different locations within the organelle, transport systems may exist for phosphatidylcholine metabolites. To test the hypothesis that rat liver microsomes contain a lysophosphatidylcholine transporter, sn- l-monobutyroylphosphatidylcholine was employed. Since this homolog is highly water-soluble, transport of lysophosphatidylcholine could be measured using standard transport methods. sn- 1-Monobutyroylphosphatidylcholine en- tered the lumenal compartment of microsomal vesicles. Transport was saturable and dependent on time and on amount of microsomes and required an intact permeability barrier. sn-1-Monobutyroylphosphatidylcholine transport was inhibited by treatment of microsomes with trypsin, N-ethylmaleimide, and trinitrobenzene- sulfonic acid. These findings suggest that sn-l-monobutyroylphosphatidylcholine transport is protein-mediated. sn- 1-Monobutyroylphosphatidylcholine transported into microsomes was degraded to glycerophosphorylcholine. Glycerophosphorylcholine was also transported across the microsomal membrane. Glycer- ophosphorylcholine transport was also saturable and dependent on time, amount of microsomes, and an intact permeability barrier but was not inhibited by treatment with trypsin or the two protein modification agents. Thus, separate and distinct transport systems exist for phosphatidylcholine metabolites. Molecular events of phosphatidylcholine turnover in the endoplasmic reticulum are discussed.

Phospholipid biosynthesis occurs asymmetrically on the cytoplasmic leaflet of the endoplasmic reticulum bilayer (1). The transmembrane movement of phospholipid to the lumenal leaflet of the endoplasmic reticulum is, therefore, an essential step of membrane assembly (1). Recently, evidence was provided that suggested that the rapid transmicrosomal

* This research was supported by Grant AM 20205 from the Na- tional Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930- 01, Japan.

J To whom correspondence should be addressed.

movement of PC‘ is protein-mediated (2). In this study, the transmicrosomal movement of the PC metabolites, lysoPC and GPC, was investigated. The movement of these com- pounds across the endoplasmic reticulum bilayer may play a central role in regulating PC metabolism.

lysoPC is a ubiquitous component of cellular membranes (3) and serves as an intermediate in the Lands’ pathway of PC synthesis (4, 5 ) . The concentration of lysoPC must be regulated, since lysoPC can cause perturbation of membrane structure (6).

Knowledge of lysoPC transmembrane movement and dis- tribution is limited (7). However, lysoPC and PC appear to behave similarly in the systems examined. The rate of IysoPC movement across artificial membranes was extremely slow with a half-time of 100 h or more (8,9). In erythrocyte plasma membranes, the half-time of lysoPC transmembrane movement ranged from 4 to 11 h (10). On the other hand, Van den Besselaar et al. (11) suggested that lysoPC moved rapidly across sarcoplasmic reticulum membranes with a half-time of less than 30 min. This rate was comparable to that of PC in sarcoplasmic reticulum (a half-time of less than 10 min) (12).

In hepatic microsomes, lysoPC accounts for 2% of the total phospholipid (3); however, data concerning its distributional asymmetry or rate of transmembrane movement are lacking. Rapid translocation of PC in rat liver microsomes has been demonstrated (13,14). Recently, Bishop and Bell (2) provided evidence that PC translocation across microsomal membranes is facilitated by a specific transport protein(s), a PC “flippase.” A PC homolog, diC4PC, which is soluble in monomeric form up to 80 mM (15) was used as a substrate for the putative flippase. This substrate retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles. This assay made it possible to determine the rate of PC transmembrane movement in 1-2.5 min (2). The similar transmembrane movement rates of PC and lysoPC in all systems examined suggest that similar mechanisms may control their movement. In partic- ular, these findings suggest that transmembrane movement of lysoPC in biogenic membranes such as hepatic microsomes may be facilitated by a specific transporter.

In the present study, monoC4PC was used as a substrate to assess lysoPC movement across microsomal membranes. The transport of monoC4PC into rat liver microsomal vesicles was demonstrated. Evidence that monoC,PC transport was protein-mediated was obtained. The uptake of monoC,PC and GPC into microsomes was compared, and evidence demonstrating that GPC is transported into microsomes by a mech-

The abbreviations used are: PC, phosphatidylcholine; IysoPC, lysophosphatidylcholine; diC,PC, dibutyroylphosphatidylcholine; monoC4PC, sn-1-monobutyroylphosphatidylcholine; GPC, sn-glycerophosphorylcholine; glycerol-P, sn-glycero-3-phosphate; TNBS, tri- nitrobenzenesulfonic acid; NEM, N-ethylmaleimide; DIDS, 4,4’-di- isothiocyanostilbene-2,2’-disulfonic acid.

16495

16496 Endoplasmic Reticulum Transporters

anism distinct from that for monoC4PC was obtained. A model for the participation of these transporters in PC turnover in hepatic endoplasmic reticulum is presented.

EXPERIMENTAL PROCEDURES

Materials-Carrier-free [32Pi], ['H]glycerol, and [n~ethyl-~H]choline were purchased from Du Pont-New England Nuclear. ["P] Glycerol-P was a generous gift from Tommy Tillman in our labora- tory. diC4PC and dioleoylphosphatidic acid were obtained from Avanti Polar Lipids. GPC (free base), snake venom (Crotulus &a- manteus), soybean trypsin inhibitor, bovine serum albumin, and protein modification reagents were from Sigma. Trypsin was from Miles Laboratories (Republic of South Africa).

monoC4PC was prepared from diC4PC by the action of snake venom phospholipase Az essentially according to Lands and Merkl (16) with the following modifications. Chloroform solution of diC4PC (20 pmol) was dried under nitrogen in a glass tube. Four ml of ether and 40 pl of snake venom solution (5 mg of snake venom/ml of 0.1 M Tris-HCl (pH 7.5) containing 10 mM CaC12) were added to the tube and vortexed well for 30 s. The reaction mixture was incubated at room temperature for 1 h with occasional vortexing (intervals of 5 min). After drying with nitrogen, the residue was dissolved in chloroform/methanol (l:l, v/v), and monoC4PC was purified by column chromatography on Bio-Si1 A (Bio-Rad). monoC4PC was eluted from the column by gradually increasing the concentration of methanol in chloroform. monoC4PC was eluted with chloroform/methanol (2:8 to 1:9, v/v). The purified monoC4PC was dried, dissolved in water, and passed through a Millipore HA 0.45 filter to remove silica gel particles. The filtrate was lyophilized, and the residue was kept in methanol at -20 "C.

Preparation of Radioactiue Substrate~--[~~P]GPC was prepared by mild alkaline hydrolysis (17) of ["PIPC prepared as described previously (21, and a small amount of [32P]glycerol-P formed during the mild alkaline hydrolysis was removed by passing through a column of Dowex 1. The final [32P]GPC product was dried under reduced pressure and stored in methanol at -20 "C. [32P]diC4PC was synthesized using butyric anhydride essentially according to Gupta et al. (18). After removing dimethylaminopyridine on a Dowex 50W-X4 column, the eluate was taken to dryness, dissolved in chloroform, and passed through a column of Bio-Sil. [32P]diC4PC was eluted from the column by gradually increasing the concentration of methanol in chloroform. diC,PC was eluted with chloroform/methanol (28, v/v). [3H]diC4PC was prepared from [3H]dibutyroylpbosphatidic acid and choline tosylate essentially according to Rosenthal (19) and purified by column Chromatography on Bio-Sil. [3H]Dibutyroylphosphatidic acid was prepared from [3H]glycerol-P by the method of Gupta et al. (18). [3H]Glycerol-P was prepared enzymatically from [3H]glycerol by the method of Cbang and Kennedy (20).

[32P]monoC4PC and [3H]monoC4PC were prepared from the cor- responding labeled diC4PCs using snake venom as described above.

Radioactive diC4PC and monoC.,PC were taken to dryness, and the residues were dissolved in water. The aqueous solution of monoC4PC and diC4PC were passed through Millipore HA 0.45 filters and then lyophilized. The dried materials were kept in methanol at -20 "C. The filtration step was necessary to reduce background in the transport assays.

[3H]GPC was prepared by mild alkaline hydrolysis (17) of 13H] dioleoylphosphatidylcholine which had been prepared from dioleoylphosphatidic acid and [3H]choline according to Rosenthal (19). The radiolabeled probes were of greater than 99% purity. This was established by thin layer chromatography on Avicel or silica gel 60 plates as described below under "Determination of Metabolites of mono- C,PC."

Preparation of Microsomes-Female rats of Charles River CD strain, weighing 170-200 g, were used. Microsomes were prepared from livers as described by Coleman and Bell (1). Samples (25-30 mg/ml) were stored in 250 mM sucrose, 10 mM Tris-HC1 buffer (pH 7.4) at -80 "C.

Transport Assay-The transport of diC4PC was assayed as described previously (2). The transport of monoC4PC and GPC was assayed by the same manner as that of diC4PC. When tritiated substrates were used for the transport assays, the filters, with trapped microsomes, were placed in counting vials without drying and dissolved with 1 ml of 2-methoxyethanol. The solubilized samples were mixed well with 10 ml of Aquasol scintillation liquid (Du Pont-New England Nuclear), and the radioactivity was counted.

Determination of Metabolites of nonoCJ'C-Microsomes incu-

bated with [32P]monoC4PC were trapped on filters and extracted with 0.8 ml of cold water, followed by 2 ml of methanol, 0.9 ml of methanol/ water (5:4, v/v), 1 ml of chloroform, and then 1 ml of chloroform/ methanol (Ll , v/v). More than 97% of radioactivity was recovered. After removing protein through a glass wool filter, the extract was dried under nitrogen or under reduced pressure. The residue was dissolved in methanol, and metabolites were analyzed by thin layer chromatography on Avicel plates (0.25 mm; Analtech) developed in n-butyl alcohol/acetic acid/water (5:3:1, v/v) and in phenol (saturated with water)/ethanol/acetic acid (505:6, v/v), and on silica gel 60 plates (0.25 mm; Merck) developed in chloroform/methanol/water/ acetic acid (251542, v/v). Standards of [32P)monoC4PC and I"P] GPC were run simultaneously. Spots containing radioactivity were identified by autoradiography (Kodak XAR-5 film), scraped, and counted in 10 ml of Aquasol scintillation liquid.

Determination of Metabolites of GPC-The radioactive materials on the filters were extracted by the same manner as described for monoC4PC. To break the phase of the chloroform/methanol/water extract, 1.5 ml of chloroform and 1.5 ml of water were added. 95% of the radioactivity was recovered in the methanol/water phase. This phase was taken to dryness under reduced pressure, and the residue was dissolved in methanol/water (109, v/v) and analyzed by thin layer chromatography on Avicel developed in phenol (saturated with water)/ethanol/water (505:6, v/v) and in n-butyl alcohol/acetic acid/ water (5:3:1, v/v). Authentic 13*P]GPC and [32P]glycerol-P were run simultaneously. Spots were identified by autoradiography, scraped, and counted with a liquid scintillation counter.

Leakage of monoCJ'C and GPC from Egg PC Vesicles-Leakage of monoC4PC and GPC from egg PC vesicles was checked by the same manner as described previously by Bishop and Bell (2) for diC4PC.

Analytical Methods-Protein was determined by the method of Lowry e t al. (21) with bovine serum albumin as standard. Lipid phosphorus was determined by the method of Ames and Dubin (22).

RESULTS

Rationale-To test the hypothesis that the transmembrane movement of lysoPC in hepatic microsomes may be catalyzed by a specific transport protein, experiments similar to those performed with diC4PC (2) were undertaken. Specifically, the dependences of lysoPC (and GPC) transport on time, on the concentration of microsomes, and on an intact permeability barrier were examined. These experiments, in conjunction with studies on nonbiogenic membranes, allowed transport to be distinguished from other uptake phenomena (e.g. binding to membrane components) (2). In addition, the effects of trypsin and protein modification reagents were examined to test if transport was protein-mediated.

Rapid Transport of lysoPC and GPC across Microsomal Membranes-Time courses of monoC4PC, GPC, and diC,PC uptake by rat liver microsomes are shown in Fig. 1. The uptake of monoC4PC reached a steady state level after about 10 min, similar to diC4PC. In contrast, GPC uptake did not reach steady state levels until 20 min. At equilibrium, approximately the same percentage (0.35-0.496) of each substrate was taken up by the fixed amount of microsomes employed. The percentage of total monoCIPC and GPC taken up at equilibrium was constant over the concentration range 0.25- 10 mM (data not shown). The maximum uptake of monoC4PC and GPC was dependent on the amount of microsomal protein. The fraction of substrate taken up at equilibrium was a linear function of the amount of microsomal vesicles in the incubation mixture (Fig. 2). From these data on monoC4PC and GPC uptake, intramicrosomal volumes of 1.5 and 1.2 pl/ mg microsomal protein, respectively, were calculated. These values are similar to the values (0.9-1.3 pl/mg microsomal protein) obtained by Bishop and Bell (2) and Carey et al. (23).

By examining the effects of detergents and toluene, Bishop and Bell (2) showed that diC,PC transport correlated with mannose 6-phosphatase latency, thus demonstrating that diC4PC uptake by microsomes required an intact permeability barrier. Triton X-100 permeabilizes microsomal membranes

Endoplasmic Reticulum Transporters 16497

0.4

0.2

O C W

k 0.4 0 a cn 5 0.2 [r t-

8 C

0.4

0. P

C

I I 1 1

-cr -f / I I I ) 10 2 0 30

TIME (mln)

FIG. 1. monoCIPC and GPC uptake by microsomes. Micro- somes (4.8 mg of protein) were incubated at 23 "C with 1 mM monoC4PC, GPC, or diC4PC in a final volume of 1.6 ml. A t the indicated times, aliquots (200 pl) of the incubation mixture were diluted into 5 ml of 0.1 M LiCl and immediately filtered using Millipore HA 0.45 filters on an Amicon filtration manifold. The reaction tube and filter were rinsed with 2.5 ml of 0.1 M LiC1, followed by an additional 5-ml wash. A , monoCrPC; B, GPC; C, diC4PC.

1.0

0.5 W I- E 0

k o z Lz + Q: I .o

,\" 0.5

0

- A

/ 500 IOOC

MICROSOMES (/us proteln)

FIG. 2. Microsomal protein dependence of monoCIPC and GPC transport. Varying amounts of microsomes, pretreated with 4 m M diisopropyl fluorophosphate to inhibit microsomal esterases, were incubated for 30 min with 1 mM monoC4PC or GPC. A , monoC4PC; n, GPC.

(24), and treatment of microsomes with Triton X-100 caused reduction of diC4PC uptake in parallel with the expression of latent mannose 6-phosphatase (2). Pretreatment of microsomes with Triton X-100 abolished monoC,PC uptake in a concentration-dependent fashion (Fig. 3A), similar to the results seen for diC,PC transport (Fig. 3C) . The uptake of GPC was also abolished by pretreatment of microsomes with

IO0

5c

o c z 1 5 C z Q r

toc > k 2 5c

a " \ o c

t- V

I oc

5c

C 0 0.04 0.0

[TRITON x-1001 Y, v/v FIG. 3. Relationship between permeability barrier and

transport. Microsomes (0.6 mg of protein) were preincubated in the presence of the indicated concentration of Triton X-100 for 10 min. monoC,PC, GPC, or diC,PC was added to a final concentration of 1 mM, and uptake was measured after 5 min. A , monoC4PC; B, GPC; C, diC4PC. In a number of experiments, 40-50% stimulation of GPC uptake rate was observed in microsomes which had been pretreated with 0.02% Triton X-100. The reason for this stimulation is not clear.

Triton X-100 although a slightly higher concentration of Triton X-100 was required (Fig. 3B) . These results indicate that the uptake of monoC4PC and GPC by microsomes re- quires an intact membrane permeability barrier, suggesting that these substrates were translocated into the lumenal compartment of microsomes.

To confirm that the transport of monoC4PC and GPC does not occur in nonbiogenic membranes, the ability of PC vesicles to transport monoC4PC and GPC was tested. monoC4PC or GPC were trapped within large unilamellar vesicles of egg PC, and the vesicles were separated from free monoC,PC or GPC by column chromatography on Sephadex G-50. These vesicles were incubated for 1 h and then rechromatographed on the Sephadex G-50 column to measure efflux of monoC4PC or GPC. All the radioactivity eluted in the void volume with the vesicle peak (data not shown). Most monoC4PC and GPC was released from the vesicles by sonication. Moreover, when free monoC4PC or GPC was incubated with preformed PC vesicles and then chromatographed on a Sephadex G-50 column, no radioactivity was found to be associated with the vesicles (data not shown). These findings indicate that monoC4PC or GPC do not spontaneously cross the PC vesicle bilayer and do not partition into PC bilayers.

Concentration Dependence of monoC4PC and GPC Trans- port-The effects of substrate concentration on initial rates of uptake of monoC4PC and GPC were investigated (Fig. 4). The initial rates of monoC4PC and GPC uptake were both linear at substrate concentrations up to about 20 mM. The rate of uptake of both substrates was saturable at higher concentrations, suggesting that a limited number of transport


[monoC4PCJ rnM

[GPC] mM

FIG. 4. Concentration dependence of monoCIPC and GPC uptake. Microsomes (0.6 mg of protein) were incubated with varying amounts of monoC,PC or GPC as indicated. Transport was measured following a 2.5-min incubation. Data points have been corrected for 0-min controls determined at each concentration of substrate. In the case of GPC, the incubation was performed in the presence of 5 mM EDTA to prevent microsomes from aggregating. 10 mM EDTA did not affect microsomal transport activity. A, monoC4PC; B, GPC.

TABLE I Effects of chemical modification reagents on monoCJ'C and GPC

transport Microsomes (0.6 mg of protein) were preincubated with TNBS in

50 mM sodium phosphate buffer (pH 7.4) for 15 min and then incubated with 5 mM substrates for 5 min. Microsomes were isolated and washed using 250 mM sucrose, 10 mM sodium phosphate buffer (pH 7.4) and resuspended in the same sucrose buffer for these experiments. Microsomes (0.6 mg of protein) were incubated with NEM or DIDS in 50 mM Tris-HC1 buffer (pH 7.4) for 15 min and then incubated with 5 mM of substrates for 5 min for transport assays. ND, not done.

Treatments Percent transport activity remaining

monoC,PC GPC diC,PC

None 100 100 100 20 mM TNBS 52.0 107 53.9 NEM

5 mM 80.1 104 78.8 10 mM 69.0 100 63.7 20 mM 53.4 96.4 53.9

0.25 mM DIDS 80.9 66.7 ND

sites exist for both monoC,PC and GPC. Effects of Modification of Microsomal Vesicles-Effects of

three chemical modification reagents on monoC,PC and GPC transport were examined (Table I). Pretreatments of microsomes with TNBS and NEM caused inhibition of monoC4PC transport. Similar inhibition of diC4PC transport was observed. Neither TNBS nor NEM affected GPC transport. DIDS, an anion transporter inhibitor, does not inhibit diC4PC transport (2). DIDS had little effect on monoC,PC transport or GPC transport. The extent of this inhibition is small compared to that of the glucose 6-phosphate transporter (25).

Pretreatment of microsomes with trypsin decreased the rate of monoC,PC uptake to a similar extent as diC,PC uptake (Table 11). However, pretreatment of microsomes with trypsin

TABLE I1 Effects of trypsin treatment on monoCJ'C and GPC transport

Microsomes (15 mg of protein/ml) were treated with trypsin (200 Kg to 1 mg of microsomal protein) for 20 min at 23 "C in 250 mM sucrose, 10 mM Tris-HC1 buffer (pH 7.4). The reaction was stopped by the addition of a 5-fold excess (w/w) of soybean trypsin inhibitor. In the control experiments, trypsin and soybean trypsin inhibitor were premixed, and then microsomes were incubated with the premixed trypsin and trypsin inhibitor. In the control experiments, microsomes were treated with NEM in the presence of trypsin and trypsin inhibitor. These pretreated microsomes were incubated with 1 mM substrates for 5 min for the transport assays.

Treatments Percent transport activity remaining

monoC,PC GPC diC,PC

None 100 100 100 NEM 65.7 118 64.8 Trypsin 64.3 96.0 70.3 Trypsin + NEM 42.8 84.4 40.7

did not cause any change in the rate of GPC uptake. Moreover, the effects of pretreatment with trypsin and NEM on monoC4PC transport were cumulative, whereas the combined pretreatment had little effect on GPC uptake (Table 11). These results strongly suggest that GPC transport occurs by a mechanism different from either monoC4PC or diC4PC transport and that monoC4PC transport is protein-mediated and has properties similar to diC4PC transport.

Efflux of Transported Substrates-If the substrates transported into the lumen of microsomes are not bound to lumenal components or metabolized, the substrates should exit in a predictable manner when the concentration of the substrates outside of microsomes is reduced. To investigate this possi- bility, microsomes were incubated with monoC4PC, GPC, or diC4PC for 30 min, and then the incubation mixtures were diluted 10-fold by the addition of buffer. Labeled substrates effluxed at rates similar to uptake (Fig. 5). At 30 min, 85% of monoC4PC, 75% of GPC, and 90% of diC4PC had exited. These findings are essentially in accord with the expectations assuming little or no metabolism had occurred.

Metabolism of monoCJ'C and GPC-Hepatic microsomes contain lysoPC acyltransferase (4,26,27) located on the outer surface of microsomal membranes (1). Moreover, hepatic microsomes contain lysophospholipase on the lumenal surface (28). This suggests two possible metabolic fates for lysoPC: (i) conversion to 1-butyroyl-2-acyl-GPC by the action of lysoPC acyltransferase (this product may be incorporated into microsomal membranes); and (ii) hydrolysis by the lumenal lysophospholipase to form GPC. To test these possibilities, the chemical forms of substrates transported into microsomes were determined. When [32P]monoC4PC was incubated with microsomes for 30 min, approximately 83% of the radioactivity existed as monoC4PC, and about 10% of the radioactivity was GPC. The remaining 7% of the radioactivity was associated with a product that moves faster than monoC4PC and slower than PC upon thin layer chromatography on silica gel plates developed with chloroform/methanol/water/acetic acid (25:15:4:2, v/v). This metabolite could be 1-butyroyl-2-acyl- GPC (Fig. 6A).

[32P]diC4PC transported into microsomes was metabolized to a greater extent than monoC4PC. After 30 min, approximately 28 and 4% of the incorporated radioactivity existed as monoC4PC and GPC, respectively, whereas no [32P]glycero-P was detected (data not shown). These data are consistent with earlier findings; De Jong et al. (29) observed that bovine liver microsomal lysophospholipase exhibits a marked preference for short chain diacylphosphatidylcholine compared to short chain monoacylphosphatidylcholine.


I O 0

50

0 c3 100

a z w 50

8

z z - -

0

100

50

0

-1 C

0 IO 20 30 TIME (min)

FIG. 5. Exit of substrates transported into microsomes. Mi- crosomes (3.6 mg of protein), pretreated with 4 mM diisopropyl fluorophosphate, were incubated with 10 mM monoC,PC, GPC, or diC,PC. At 30 min, an aliquot (200 p1) was removed for filtration and, immediately, the incubation mixture was diluted by adding 9 volumes of buffer. At the indicated times, aliquots (1 ml) were filtered. A, monoC4PC; B, GPC; C, diC,PC.

Rat liver microsomes also contain GPC phosphodiesterase activity (30). Since little information is available about this activity in rat liver microsomes, we measured the activity in our preparations. In accordance with the preliminary findings of Baldwin and Cornatzer (30), the optimal pH for GPC phosphodiesterase in rat liver microsomes was around 9.5, and the specific activity was about 1 nmol/min/mg protein at a GPC concentration of 2.5 mM. At pH 7.4, this enzyme showed high activity (40% of the full activity at pH 9.5). Since microsomes are known to be disrupted at high pH, it is impossible to obtain information about both transport and metabolism of GPC at a pH of 9.5. At pH 7.4, about 98% of radioactivity, associated with microsomes after 30 min, existed as GPC, and the amount of glycerol-P detected was very small (about 0.8%) (Fig. 6B).

DISCUSSION

The transbilayer movement of phospholipids is extremely slow in phospholipid vesicles (31-34). Ganong and Bell (35) showed that dioleoylthioglycerol, a diacylglycerol analog, translocated rapidly across artificial vesicle membrane with a half-time of less than 15 s, whereas transmembrane movement of phosphatidylthioglycerol is extremely slow with a half-time of more than 8 days. These findings strongly suggested that the polar head group of the phospholipid molecule is the portion of the phospholipid unable to move spontaneously.

‘ 0 L IO 20 30

TIME ( r n i n ) FIG. 6. Metabolism of monoCIPC and GPC. Microsomes (3

mg of protein) were incubated with 5 mM [32P]monoC4PC in a final volume of 0.8 ml, or microsomes (3.6 mg of protein) were incubated with 2.5 mM [32P]GPC in a final volume of 1.2 ml. At the indicated times, an aliquot (200 rl) was filtered. The filters were extracted, and metabolites were analyzed by thin layer chromatography as described under “Experimental Procedures.” Data points have been corrected for a 0-min control done separately. A , monoC,PC; B, GPC. W, monoC,PC; c”., GPC; IC”. , glycerol-P; U, I-butyroyl- 2-acyl-GPC.

Therefore, it is reasonable to suggest that specific mechanisms must facilitate transmembrane movement of the phospholipid head group in membranes where rapid flip-flop occurs. Based on these findings, Bishop and Bell ( 2 ) utilized diC4PC to develop a new method to measure PC transmembrane movement in microsomes. diC,PC is water-soluble in monomeric form up to concentrations of 80 mM (15) and does not partition into the lipid bilayer of microsomal membranes. Using this new method, Bishop and Bell ( 2 ) demonstrated that diC4PC equilibrated between the external and lumenal compartments and that the translocation of diC,PC was protein- mediated.

In the present study, we used monoC,PC to study lysoPC transmembrane movement by a similar approach. monoC,PC is highly soluble in aqueous solutions, allowing transmembrane movement to be assayed by standard transport methods. Moreover, monoC4PC (unlike long chain lysoPC) does not cause perturbation of membrane integrity. monoC4PC transport was shown to be dependent on time and amount of microsomes employed (Figs. 1 and 2 ) . Transport of monoC,PC required an intact microsomal permeability barrier (Fig. 3). The fraction of monoC,PC taken up at equilibrium was a function of the amount of microsomal vesicles and was inde- pendent of the initial substrate concentration. Moreover, transport was bidirectional (Fig. 5). These results, together with the calculated lumenal volumes, suggest that monoC4PC had equilibrated between the external and internal compartments. The saturable initial rate of monoC4PC uptake suggested that a limited number of sites for monoC,PC translocation existed (Fig. 4). The inhibition of transport by NEM, TNBS, and trypsin suggests that transport is protein-mediated (Tables I and 11). The results shown in Tables I and I1 indicate that the monoC4PC transporter has properties simi-


lar to those of the diC,PC transporter.' The data are consistent with diCsPC and monoC4PC transport activities being dual functions of a single transporter. However, purification of these transporters will be necessary to conclude whether the same protein catalyzes both transport processes.

The finding of monoC4PC transport led us to investigate GPC transport in microsomes. GPC was transported across the microsomal membrane. Based on the same criteria that we used to establish monoC4PC transport, GPC equilibrates between external and lumenal compartments. GPC transport was also saturable in its initial rate, suggesting a limited number of transport sites on microsomes. I t is noteworthy that, in the case of glycerol and sucrose which are known to penetrate into microsomes by simple diffusion, the penetra- tion of both are linear up to 250 mM (23). The transport of GPC was not inhibited by treatment of microsomes with either NEM, TNBS, or trypsin. These results strongly suggest that GPC transport2 is mediated by a different transporter than that functioning to transport monoC4PC or diC4PC.

Microsomes contain a glucose 6-phosphate transporter (36) which is strongly inhibited by DIDS (25). Although DIDS was slightly inhibitory for both mohooC4PC and GPC transport (Table I), the extent of the inhibition is small compared to that of the glucose 6-phosphate transporter (25). These results indicate that both monoC4PC transport and GPC transport have properties which are different from those of glucose 6- phosphate transport. Several facilitated transport systems have now been described in rat liver microsomes. In addition to the glucose 6-phosphate transporter, the transport systems for inorganic phosphate (37), Ca'+ (38), and nucleotide sugars (23) have been identified. The nucleotide sugars transport system appears confined to the Golgi vesicle components of microsomes (39). Thus, identification of facilitated transport systems for diC4PC (2) and now monoC,PC and GPC are additions to a short list. It is likely that other transport systems for phospholipids and/or their metabolites may exist in the endoplasmic reticulum.

It is likely that the monoC4PC transporter of microsomal vesicles also functions to transport the endogenous long chain lysoPC homologs. monoC4PC is not a naturally occurring homolog, and no other physiologically relevant reason for the existence of a transporter capable of facilitating its movement in rat liver microsomes is apparent.

lysoPC is considered to be an important intermediate in the remodeling of PC (4, 5). lysoPC has been suggested to play a physiological role in the regulation of a number of membrane-associated enzymes (6). Since high concentrations of 1ysoPC cause perturbations of membrane structure (61, lysoPC levels in biogenic membranes must be strictly con- trolled. In spite of the fact that PC turns over rapidly (40), the concentration of lysoPC in microsomes of rat liver is maintained at about 2% (3).

Two possible mechanisms exist by which microsomes may control IysoPC concentrations, reacylation to PC or degradation to GPC and other metabolites. Hepatic microsomes contain 1-acyl-GPC:acyl-CoA acyltransferase and 2-acyl- GPC:acyl-CoA acyltransferase with very high specific activities (26, 27, 41). These acyltransferases have been shown to contribute greatly to remodeling of de mu0 synthesized PC (5, 42, 43). However, the contribution of these enzymes to scavenge lysoPC is unknown. Other workers suggest that the fate of lysoPC may be further degradation to water-soluble metabolites. For example, Bjornstad (44) provided evidence

Human red blood cells did not take up monoC4PC or GPC when tested as described earlier for diCrPC (2); diC4PC was shown earlier not to enter red blood cells (2).

that endogenous PC was degraded to acid-soluble metabolites (35% GPC and 61% choline) without significant accumulation of lysoPC. These findings clearly showed that rat liver microsomes contain a PC turnover pathway involving phospholi- pases A1 and Az, lysophospholipase, and GPC phosphodiesterase.

Two lysophospholipases have been purified from bovine liver (45). De Jong et al. (29) showed that one was localized in both cytosol and mitochondria and the other was localized in microsomes. Subsequently, the microsomal enzyme was shown to be located at the lumenal surface of the microsomal vesicle (28). Although, in rat liver, the bulk of lysophospholipase activity was recovered in the cytosolic fraction, considerable activity was detected in microsomes (46-48). Our preliminary determination of lysophospholipase activity in rat liver microsomes gave about 1 nmol/min/mg of microsomal protein when lysoPC was used as a sub~t ra te .~ This activity is comparable to the values obtained by Lumb and Allen (49) and Loffler et al. (48) and to the activities of microsomal phospholipase A, and Az (49-51).

As shown in Fig. 7, transporters for lysoPC and GPC may play a central role in lysoPC metabolism. lysoPC may be formed by the action of microsomal phospholipase AI and A2 (40, 52). Recently, Beckman et al. (51) provided suggestive data about the intramicrosomal localization of these phospho- lipases in rat liver; they showed that phospholipase A, could be assayed using a labeled substrate in liposomes, whereas phospholipase Al activity was not detected using the liposome substrate but could be detected using labeled endogenous substrates present in microsomes. These findings suggested a localization of phospholipase A, on the cytoplasmic side of microsomes. lysoPC formed on the cytoplasmic surface of microsomes by phospholipase A, may be translocated by the lysoPC transporter to the lumenal leaflet of microsomal membranes for further degradation by the lumenal lysophospholipase to GPC. Alternatively, lysoPC formed on the lumenal side by phospholipase AB may be transported to the cytoplasmic side where it can be reacylated by the action of 1ysoPC acyltransferase.

GPC, formed in the lumen, may be further degraded by GPC phosphodiesterase. Most tissues of rat contain GPC phosphodiesterase which catalyzes the formation of glycerol- P and choline from GPC (30, 53). This enzyme is localized in microsomes in rat kidney (30). Rat liver microsomes contain considerable GPC phosphodiesterase activity (about 1-3 nmol/min/mg protein)? At present, there is no data available about the topography of GPC phosphodiesterase in microsomes. This enzyme is likely to be localized at the cytoplasmic surface of microsomes, since little glycerol-P was detected as a metabolite of lumenal diC4PC, monoC,PC, or GPC. Thus, GPC formed in the lumenal compartment may be translocated to the outer surface of microsomes where it may be degraded by GPC phosphodiesterase to glycerol-P and choline: This process would allow for glycerol-P and choline to be recycled. "

Y. Kawashima and R. M. Bell, unpublished data. The apparent K, for monoCIPC and GPC transport is greater

than 10 mM. The high apparent K , for diC4PC was discussed previously (2). The use of a water-soluble analogue of PC, diC,PC, and the

portant considerations. These considerations would extend to IysoPC high membrane-bound endogenous microsomal PC content are im-

and monoC4PC. However, GPC would be soluble; cellular concentrations of GPC have not been established. If GPC is produced within the microsomal lumen, a high lumenal content could be achieved; the high K,,, could, therefore, regulate effectively the exit of GPC into the larger cytoplasmic volume. Further studies are needed to address the specificity of the GPC transporter; it is possible that this activity is a side reaction of a transporter whose physiological function is for another phosphodiester-containing metabolite.


LUMEN

FIG. 7. Possible turnover pathway of phosphatidylcholine in hepatic microsomes. 1 , PC flippase; 2, lysoPC flippase; 3, GPC transporter; 4 , phospholipase AI and AZ; 5, lysophospholipase; 6, glycerophosphorylcholine phosphodiesterase.

The results of this study suggested that the transporters for lysoPC and GPC may play a crucial role in regulating microsomal lysoPC concentration and metabolism.

Acknowledgments-We thank Drs. W. Robert Bishop and Carson Loomis for their helpful discussions. We also thank Dr. W. Robert Bishop for performing the studies on monoCIPC and GPC uptake into human red cells.

REFERENCES

1. Coleman, R., and Bell, R. M. (1978) J. Cell Biol. 76, 245-253 2. Bishop, W. R., and Bell, R. M. (1985) Cell 42,5-60 3. White, D. A. (1973) in Form and Function of Phospholipids

(Ansell, G. B., Hawthrone, J. N., and Dawson, R. M. C., eds) pp. 441-478, Elsevier, New York

4. Lands, W. E. M. (1965)Annu. Rev. Biochem. 34,313-346 5. Houlb, B. J., and Kuksis, A. (1978) Adv. Lipid Res. 16 , 1-125 6. Weltzien, H. U. (1979) Biochim. Biophys. Acta 559,259-287 7. Op den Kamp, J . A. F. (1979) Annu. Reu. Biochem. 48,47-71 8. De Kruijff, B., Van den Besselaar, A. M. H. P., and Van Deenen,

9. Van den Besselaar, A. M. H. P., Van den Bosch, H., and Van Deenen, L. L. M. (1977) Biochim. Biophys. Acta 465 , 454-465

10. Bergmann, W. L., Dressler, V., Haest, C. W. M., and Deuticke, B. (1984) Biochim. Biophys. Acta 772 , 328-336

11. Van den Besselaar, A. M. H. P., De Kruijff, B., Van den Bosch, H., and Van Deenen, L. L. M. (1979) Biochim. Biophys. Acta 555, 193-199

12. De Kruijff, B., Van den Besselaar, A. M. H. P., Van den Bosch, H.. and Van Deenen. L. L. M. (1979) Biochim. Biophys. Acta

L. L. M. (1977) Biochim. Biophys. Acta 465,443-453

555, 181-192 . .

13. Zilversmit, D. B., and Hughes, M.E. (1977) Biochim. Biophys. Acta 469, 99-110

14. Van den Besselaar, A. M. H. P., De Kruijff, B., Van den Bosch, H., and Van Deenen, L. L. M. (1978) Biochim. Biophys. Acta 510,242-255

15. Wells, M. A. (1974) Biochemistry 13,2248-2257 16. Lands, W. E. M., and Merkl, I. (1963) J. Biol. Chem. 238 , 898-

904 17. Kates, M. (1972) in Techniques of Lipidology (Work, T. S., and

Work, E., eds) pp. 558-559, Elsevier Scientific Publishing Co., Inc., New York

18. Gupta, C. M., Radhakrishman, R., and Khorana, H. G. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,4317-4319

19. Rosenthal, A. F. (1975) Methods Enzymol. 14,429-529 20. Chang, Y-Y., and Kennedy, E. P. (1967) J. Lipid Res. 8,447-455 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

22. Ames, B. N., and Dubin, D. T. (1960) J. Biol. Chem. 235 , 769- (1951) J. Biol. Chern. 193, 265-275

775 23. Carey, D. J., Sommers, L. W., and Hirschberg, C. B. (1980) Cell

19,597-605 24. Polokoff, M. A., and Bell, R. M. (1978) J. Biol. Chem. 253,7173-

25. Zoccoli, M. A., and Karnovsky, M. L. (1980) J. Biol. Chem. 255,

26. Lands, W. E. M., and Hart, P. (1965) J. Biol. Chern. 240,1905-

27. Eibl, H., Hill, E. E., and Lands, W. E. M. (1969) Eur. J. Biochem.

28. Moonen, J. H. E., and Van den Bosch, H. (1979) Biochim.

29. De Jong, J. G. N., Dijkman, R., and Van den Bosch, H. (1975)

30. Baldwin, J. J., and Cornatzer, W. E. (1968) Biochim. Biophys.

7178

1113-1119

1911

9, 250-258

Biophys. Acta 573, 114-125

Chem. Phys. Lipids 15 , 125-137

Acta 164, 195-204

16502 E ~ o ~ ~ r n i c Reticulum Transporters 31. Rothman, J. E., and Dawidowicz, E. A. (1975) Biochemistry 14,

32. Johnson, L. W., Hughes, M. E., and Zilversmit, D. B. (1975)

33. Roseman, M., Litman, B. J., and Thompson, T . E. (1975) Bio-

34. Dicorleto, P. E., and Zilversmit, D. B. (1979) Biochim. Biophys.

35. Ganong, B. R., and Bell, R. M. (1984) Biochemistry 23, 4977-

36. Ballas, L. M., and Arion, W. J. (1977) J. Biol. Chem. 252,8512-

37. Arion, W. J., Lange, A. J., Walls, H. E., and Ballas, L. M. (1980)

38. Moore, L., Chen, T., Knapp, H. R., Jr., and Landon, E. J. (1975)

39. Sommers, L. W., and Hirschberg, C. B. (1982) J. Biol. Chem.

40. Van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246 41. Kawashima, Y., Hirose, A., and Kozuka, H. (1984) Biochim.

2809-2816

Biochim. Biophys. Acta 375, 176-185

chemistry 14,4826-4830

Acta 552, 114-119

4983

8518

J. Biol. Chem. 255,10396-10406

J. Bwl. Chem. 250,4562-4568

257,10811-10817

Biophys. Acta 793,232-237

42. Kanoh, H., and Akesson, B. (1977) Biochim. Biophys. Acta 486,

43. Kawashima, Y., Hirose, A., Adachi, T., and Kozuka, H. (1985)

44. Bjornstad, P. (1966) Biochim. Bwphys. Acta 116, 500-510 45. De Jong, J. G. N., Van den Bosch, H., Rijken, D., and Van

Deenen, L. L. M. (1974) Biochim. Biophys. Acta 369, 50-63 46. Van den Bosch, H., Aarsman, A. J., Slotboom, A. J., and Van

Deenen, L. L. M. (1968) Biochim. Biophys. Acta 164, 215-225

511-523

Biochim. Biophys. Acta 837, 222-229

47. Leibovizt-Ben Gershon, Z., and Gatt, S. 11976) Biochem. Biophys.

48. Loffler. B-M.. Bohn. E.. Hesse. B.. and Kunze. H. (1985) Biochim. Res. Commu~. 69,592-598

Biophys, Acta 835,448-455 '

, . -

49. Lumb. R. H.. and Allen. K. F. (1976) Biochim. Bionhvs. Acta 450, 175-184

50. Momchilova, A., Petkova, D., and Kumanov, K. (1986) Znt. J . Biochem. 18, 659-663

51. Beckman, J. K., Borowitz, S. M., and Burr, I. M. (1987) J. Biol. Chem. 262, 1479-1481

52. Van den Bosch, H. (1982) in P ~ s ~ h o l ~ p ~ s ( H a ~ h r o n e , J. N., and Ansell, G. B., eds) pp. 313-357, Elsevier Scientific Publish- ing Co., Amsterdam

"

53. Dawson, R. M. C. (1956) Biachem. J. 62,689-693

Documents

Assembly of the Endoplasmic Reticulum Phospholipid Bilayer