Biosynthesis of Glycerolipid Precursors in Rat Liver Peroxisomes and Their Transport and Conversion to Phosphatidate in the Endoplasmic Reticulum*

The transport of glycerolipid intermediates, uiz. pal- mitoyl dihydroxyacetone phosphate (DHAP) and lysophosphatidate from peroxisomes and their conversion to phosphatidate in endoplasmic reticulum (micro-somes) were studied in cell-free systems. The lipids were biosynthesized from [s2P]DHAP, palmitoyl-CoA, and freshly made rat liver peroxisomes and micro- somes in the presence or absence of M8+, NADPH, and bovine serum albumin (BSA). After incubation, the soluble fraction and the membranes were separated, and the distribution of radioactive lipids in these frac- tions were determined. The results showed that pal-mitoyl-DHAP and lysophosphatidate were recovered in the supernatant when BSA was present or when BSA was absent, but Mg2+ was removed after incuba- tion by chelation with EDTA (or ATP). At low optimum palmitoyl-CoA concentration or when palmitoyl-CoA was generated in peroxisomes, and in the absence of BSA, the biosynthesized keto ether and ester lipids and lysophosphatidate were similarly present in the supernatant. Phosphatidate, however, was always localized in the membranes. Further fractionation showed that phosphatidate was associated with the microsomes. The critical micellar concentrations of palmitoyl- DHAP and

The transport of glycerolipid intermediates, uiz. palmitoyl dihydroxyacetone phosphate (DHAP) and lysophosphatidate from peroxisomes and their conversion to phosphatidate in endoplasmic reticulum (microsomes) were studied in cell-free systems. The lipids were biosynthesized from [s2P]DHAP, palmitoyl-CoA, and freshly made rat liver peroxisomes and microsomes in the presence or absence of M 8 + , NADPH, and bovine serum albumin (BSA). After incubation, the soluble fraction and the membranes were separated, and the distribution of radioactive lipids in these fractions were determined. The results showed that palmitoyl-DHAP and lysophosphatidate were recovered in the supernatant when BSA was present or when BSA was absent, but Mg2+ was removed after incubation by chelation with EDTA (or ATP). At low optimum palmitoyl-CoA concentration or when palmitoyl-CoA was generated in peroxisomes, and in the absence of BSA, the biosynthesized keto ether and ester lipids and lysophosphatidate were similarly present in the supernatant. Phosphatidate, however, was always localized in the membranes. Further fractionation showed that phosphatidate was associated with the microsomes. The critical micellar concentrations of palmitoyl-DHAP and 1-palmitoyl-rac-glycerol 3-phosphate, under the incubation conditions used, were determined to be 68 and 70 @M, respectively. These results suggest that at physiological concentrations the biosynthesized lysolipids are water soluble, and therefore, a carrier protein is unnecessary for their transport. These lipids freely diffuse from peroxisomes to endoplasmic reticulum where they are converted to membrane-bound phosphatidate.
In eukaryotes, enzymes catalyzing glycerolipid biosynthesis are present mainly in mitochondria and the endoplasmic reticulum (ER)' (Wilgram and Kennedy, 1963;Van den Bosch, 1974;Bell and Coleman, 1980;Hajra et al., 1986). The main site of cellular glycerolipid biosynthesis is believed to be the ER, from which the lipids are transported to different subcellular compartments (Kennedy, 1986;Bishop and Bell, 1988;Simoni, 1988). The enzyme composition of mitochondria suggests that only certain mitochondria-specific lipids (e.g. cardiolipin) are biosynthesized in these organelles (Kiyasu et al., 1963;Haldar et al., 1983). Glycerolipids and glycerol-ether lipids are also shown to be biosynthesized in animal peroxisomes . These organelles have been shown to contain the enzymes of the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway, namely DHAP acyltransferase, alkyl-DHAP synthase, and acyl/alkyl-DHAP reductase Hajra and Bishop, 1982). DHAP acyltransferase, which catalyzes the biosynthesis of acyl-DHAP, has been shown by different workers, to be localized exclusively on the inner face of the peroxisomal membrane (Rock et al., 1977;Jones and Hajra, 1979;Hardeman and Van den Bosch, 1988). Similarly, alkyl-DHAP synthase, which catalyzes the formation of ether bond, is localized inside the peroxisomal membrane Hardeman and Van den Bosch, 1988). However, acyl/ alkyl-DHAP reductase, which catalyzes the formation of lysophosphatidate (and its ether analog) is localized on the outside (cytosolic side) of the peroxisomal membrane (Ghosh and Hajra, 1986a). This reductase is also present in the ER (Ghosh and Hajra, 1986a). A fourth lipid biosynthetic enzyme, acyl-CoA reductase (long chain alcohol-forming), has recently been localized to be present on the outside of peroxisomes (Burdett et al., 1991). Most other known glycerolipid-biosynthesizing enzymes are absent in peroxisomes Ballas et al., 1984). From the composition and topography of peroxisomal enzymes, it seems that acyl-DHAP and alkyl-DHAP synthesized inside the peroxisomes are exported out and then reduced to 1-acyl and l-alkyl-glycerol-3-P, respectively, by acyl/alkyl-DHAP reductase (and cytosolic NADPH) Hardeman and Van den Bosch, 1989). The lysophosphatidate, i.e. LPA, thus formed is transported to the ER where it is converted to phosphatidate, the precursor of all glycerolipids (Kennedy, 1986). Therefore, it is evident that only glycerolipid biosynthetic intermediates are synthesized in peroxisomes and are transported to the ER to form membrane glycerolipids and triglycerides.
The mechanism of intracellular transport of water-insoluble lipids is poorly understood. It is believed that such transport is mediated either by carrier proteins or by vesicles (Zilversmit, 1984;Simoni, 1988). However, the lipids synthesized in peroxisomes contain a single acyl (or alkyl) chain and are probably water soluble (high critical micellar concentrations, CMC) at physiological concentrations (cO.1 mM, see Hajra, 1984, 1989). Therefore, it is assumed that these lipids could freely diffuse from peroxisomes to ER without any special transport mechanism . We have previously provided preliminary evidence for such a mechanism by showing that in a cell-free system the acyl-DHAP and LPA biosynthesized in peroxisomes are present in the soluble fraction, whereas phosphatidate synthesized in the ER is present in membrane-bound fractions (Horie and Hajra, 1987;Hajra et al., 1988). However, the results reported by Hardeman and van den Bosch (1989) indicate that bovine serum albumin (BSA) present in the reaction mixture acts as a carrier protein which extracts acyl-DHAP or alkyl-DHAP from peroxisomes to the supernatant. In the absence of BSA, all of acyl/alkyl-DHAP remained associated with the peroxisomal membrane (Hardeman and Van den Bosch, 1989). A similar study by Haldar and Lipfert (1990) on the mitochondrial biosynthesis of LPA also indicates that BSA is necessary for the transport of LPA from mitochondria to the ER. Therefore, it seems that a putative carrier protein might be involved in the transport of lyso lipids from one subcellular compartment to another. In this paper we reexamine this problem regarding the formation of acyl-DHAP and LPA in peroxisomes and their in vitro transport to the ER where they are converted to phosphatidate. The roles of BSA and other cofactors, especially Mg+, in this transport process are also investigated.

Methods
Isolation of Peroxisomes and Microsomes from Rat Liver-Subcellular fractionation of rat liver by differential centrifugation was done with minor modifications, as described previously (deDuve et al., 1955;Hajra et al., 1979). Briefly, the liver from adult rats was homogenized in ice-cold buffer containing 0.25 M sucrose, 10 mM TES, pH 7.5, 1 mM EDTA, 0.1% ethanol, 0.4 mM phenylmethylsulfonyl fluoride, and 0.2 mM leupeptin. The whole homogenate was subjected to differential centrifugation. The nuclear and mitochondrial pellets obtained at 600 X g for 10 min and 3,300 X g for 10 min, respectively, were discarded. The light mitochondrial fraction (L) sedimented at 25,000 X g for 10 min was resuspended in the above sucrose-TES-EDTA buffer in a volume which corresponded to 1 ml/ g wet weight of original liver tissue. Peroxisomes were isolated from this L-fraction by the method described by Ghosh and Hajra (198613) with modifications: 2 ml of the L-fraction was layered over 10 ml of 30% Nycodenz (w/v) containing TES (10 mM, pH 7.5) and EDTA (1 mM) and centrifuged at 25,000 rpm (56, 800 X gav) for 15 min in a L8-70 Beckman ultracentrifuge using a Ti-50.2 rotor. This step of peroxisome preparation using 30% Nycodenz was repeated once more to further purify this organelle. The final peroxisomal pellet was suspended in the above homogenizing medium in a volume equivalent to one-fourth of the original liver weight. Microsomes from the post L-supernatant were sedimented at 100,000 X g for 60 min and suspended in the homogenizing medium in a volume corresponding to 1 ml/g of liver. These preparations of peroxisomes and microsomes were used fresh for the study of biosynthesis and transport of lipids. The remaining portions were stored at -20 "C for assaying marker enzymes (described below). The isolated peroxisomes and microsomes, when assayed for NADPH-cytochrome c reductase (microsomal marker enzyme, Williams and Kamin, 1962), were found to have activities of 4-9 nmol/min/mg protein for the peroxisomal fraction and 150 nmol/min/mg protein for the microsomal fraction. Calculations based on these marker enzyme activities indicate that the peroxisomes were contaminated by 3-6% of microsomal protein. Mitochondrial contamination (determined by its marker succinatecytochrome c reductase, Schnaitman and Greenawalt, 1968) in this isolated peroxisomes was about 2-5% (see also Ghosh and Hajra, 1986a). The purity of peroxisomes was calculated to be 90-95%. The specific activity of DHAP acyltransferase (DHAPAT) in peroxisomes was found to be 12.0 nmol/min/mg protein under optimum conditions of assay . The enrichment of DHAPAT from post nuclear supernatant to L-fraction was about 6-fold and from the L-fraction to peroxisomes was 7-fold. Between these two latter fractions, the specific activity of catalase, the marker enzyme for peroxisomes, was also found to be increased to 7-8-fold (260 * 20 and 1800 f 300 units/mg protein in L and peroxisomes, respectively). Specific activity of DHAPAT in isolated microsomes was 0.45 nmol/min/mg protein.
Biosynthesis of Lipids-Enzymatic syntheses of acyl-DHAP, LPA, and PA were studied using fresh liver peroxisomes and microsomes by the method described previously . The incubation mixture in 2.4 ml contained Tris-HC1 (75 mM, pH 7.5), MgC12 ( BSA (4 mg), and liver microsomes (200 pg of protein) were also present in the incubation mixtures. The reaction mixtures were incubated for 30 min at 37 "C. Lipids from the reaction mixtures were extracted by the method of Bligh and Dyer (1959) under acidic condition (Hajra, 1974). An aliquot of the CHCl, solution containing the products was used to determine the radioactivity, and the remainder was used to characterize the products by silica gel TLC using a solvent mixture of CHC13/methanol/acetic acid/5% Na-metabisulfite (1004012:4) (Hajra, 1968). The radioactive spots were localized by autoradiography and characterized with respect to the Rf values of the known standards on the plate. In most experiments, three distinct and well separated 32P-labeled lipids were found to be present on the radioautograms having R, values of 0.20, 0.35, and 0.72 corresponding to those of the standard samples of palmitoyl-DHAP, LPA, and PA, respectively. The radioactive spots were scraped off into liquid scintillation minivials, mixed with 0.5 ml of HzO by sonication, and the radioactivities were determined in a liquid scintillation counter after adding 2 ml of scintillation fluid (Universol, ICN Biomedicals, Inc., Irvine, CA).
Separation of Peroxisomes and Microsomes after Incubations-A reaction mixture containing peroxisomes, microsomes, and other ingredients was immediately cooled on ice for 5 min after incubation, mixed with an equal volume of cold sucrose-EDTA (0.25-0.01 M) solution whenever needed, and then subjected to centrifugation (+4 "C) at 8,000 rpm (7,720 X g) for 10 min in a Sorval RC2-B centrifuge using a SS-34 rotor to sediment peroxisomes. The supernatant was centrifuged at 40,000 rpm (100,000 X g ) for 90 min using a Ti-50.2 Beckman rotor to sediment microsomes. Radioactive lipids from peroxisomal pellet, microsomal pellet, and from an aliquot of the final supernatant were isolated as described above. Radioactivity in total phospholipids and that of individual class, i.e. palmitoyl-DHAP, LPA, and PA present as membrane bound and in the supernatant (after centrifugation), were then determined by separating the lipid products on silica gel TLC plate followed by radioautography and then determining the ,' P counts as above.
Other Methods-Protein was determined using BSA as standard by Lowry assay procedure (Lowry et al., 1951) after it was coprecipitated with deoxycholic acid (Bensadoun and Weinstein, 1976) to remove interfering Nycodenz from the sample. The amount of l-["C] hexadecyl DHAP formed in peroxisomes from long chain 1-[14C] hexadecanol was measured radiometrically employing a solvent partition method at high pH as described by Davis and Hajra (1981). The nature of the radioactive product was characterized by TLC (CHC13/methanol/acetic acid/water, 100:40:12:4) which showed the presence of only one "C-labeled spot (Rf = 0.34) corresponding to the standard hexadecyl DHAP (Hajra et al., 1983). NADPH-cytochrome c reductase (the microsomal markers), succinate-cytochrome c reductase (the mitochondrial marker), and catalase (the peroxisomal marker) were assayed following the methods of Williams and Kamin (1962), Schnaitman and Greenawalt (1968), and Peters et al. (1972), respectively.

Effect of BSA on the Biosynthesis and Localization of Lip-
ids-Studies were carried out to investigate the role of BSA in the biosynthesis of glycerolipids intermediates. Table I shows the formation of acyl-DHAP, LPA, and PA in peroxisomes or microsomes or in combinations of them under different experimental conditions, such as in the presence or absence of NADPH and of BSA. As shown, palmitoyl-DHAP is formed mainly in peroxisomes. Most of this keto lipid was reduced (95-97%) to palmitoyl glycerol-3-P (i.e. LPA) in this organelle when NADPH was present in the incubation mixture. When the incubation mixture contained microsomes in addition to peroxisomes and NADPH, PA was the major product. Since the peroxisomes and microsomes used were not completely free from each other (see "Experimental Procedures"), a small fraction of the total lyso-PA formed in the presence of NADPH was converted to PA when peroxisomal fraction was used (Table I). Similarly, a small amount of palmitoyl-DHAP was formed when microsomes were used and was ultimately transformed into PA in the presence of NADPH (Table I). The effect of BSA on peroxisomal and microsomal lipid biosynthesis revealed that although BSA stimulated the formation of the lipids, it was not required if M$+ was present in the incubation mixture. However, BSA stimulated overall lipid production by 2.5-3 times (Table I).
In a separate experiment, the effect of Mg2+ on peroxisomal DHAPAT in the absence of BSA was investigated. The specific activities of the enzyme were 3.4 nmol/min/mg protein and 0.26 nmol/min/mg protein in the presence or absence of M$+, respectively.
The effects of BSA on the localization (membrane-bound or soluble) of acyl-DHAP, LPA, and PA formed are shown in Fig. 1. Peroxisomes and microsomes were sedimented together by centrifuging the mixture at 100,000 X g for 90 min at 4 "C after the end of incubation and the type and amount of lipids in the membrane pellet and the supernatant were determined. As shown in Fig. lA, palmitoyl-DHAP and LPA produced in peroxisomes in the presence of BSA were mostly recovered in the supernatant. In contrast, when BSA was absent but M e was present, these lipids remained mainly in the membrane fraction (Fig. 1B). However, since the incubation mixture contained M e , therefore, it is probable that these lysolipids which should be water soluble at these low concentrations (high CMC, see "Appendix") probably formed insoluble salts with divalent Mf and remained with the membrane fraction in the absence of BSA. To verify this, Mg2+ in the mixture (no BSA) was chelated by adding EDTA at the end of incubation, and the distribution of radioactive lipids were determined. The results showed that under such conditions, both acyl-DHAP and LPA were present in the supernatant even though BSA was absent in the incubation mixture (Fig. 1C). The results also showed that when PA was formed in the presence of microsomes (and NADPH), it remained bound to the membrane fraction regardless of whether BSA or EDTA was present or absent in the system (Fig. 1, A X ) .
These results show that when Mf in the incubation mixture was removed by EDTA at the end of incubations, acyl-DHAP and LPA became soluble and did not sediment with the membrane fraction. Intracellular M$+ is present mostly in the chelated form with cellular ATP (Lehninger, 1982). When ATP was added to the incubation mixture (no BSA), most (>75%) of acyl-DHAP and LPA were present in the supernatant (data not shown, but see Table 11) similar to the case when EDTA was added.
Localization of Membrane-bound PA-The results in Fig. 1, A-C, showed that the PA once formed was recovered with the membrane pellet which consisted of both peroxisomes and microsomes. To determine whether or not PA synthesized in microsomes remained exclusively bound with this organelle, peroxisomes and microsomes were separated from each other after the end of incubation. When such an experiment was performed following conventional differential centrifugation, i.e. 25,000 X g for 10 min to sediment peroxisomes and 100,000 X g for 60 min to sediment microsomes, it was observed that a large (50-60%) fraction of microsomes (as determined by the marker enzyme NADPH-cytochrome c reductase) sedimented with peroxisomes. Since the incubation mixture for PA biosynthesis contained M e , the microsomes probably aggregated in the presence of this divalent cation (Schenkman and Cinti, 1978) and sedimented at lower centrifugal force. Optimum conditions were developed to separate microsomes (ER vesicles) from peroxisomes after binding the M$+ with EDTA at the end of the reaction. The best separation was
For biosynthesis of alkyl-DHAP, the same mixture as above was used except that the pH of the buffer was 8.0, DHAP was non-radioactive, and l-['4C]hexadecanol (33.3 p~, 60,000 cpm/nmol) was included. The incubations (37 "C for 30 min) were carried out either in the absence or presence of BSA (2 mg). At the end of incubation the mixtures were cooled in ice and centrifuged at 25,000 g X 15 min at 4 "C to sediment the peroxisomes. The amount of a~yl-[~'plDHAP present in the pellet and supernatant was determined by acid extractions and washing and the amount of [14C]alkyl-DHAP formed in these fractions was determined by a solvent partition as described under "Experimental Procedures." The percentages of radioactive lipid product present in each fraction are shown. The average total amount of acyl-DHAP and alkyl-DHAP formed was 0.97 and 0.18 nmol, respectively. achieved by centrifuging the mixtures at 7,720 X g for 10 min to sediment peroxisomes and 100,000 x g for 90 min to sediment microsomes. However, even under such conditions peroxisomal fraction contained a significant amount of endoplasmic reticulum vesicles as determined by measuring the NADPH-cytochrome c reductase activity in these isolated fractions. The activity of the reductase was 4-9 nmol/min/ mg protein in the original peroxisomes and 150-160 nmol/ min/mg in the original microsomes uersus 40-50 nmol/min/ mg protein in the reisolated peroxisomal fraction and 150-160 nmol/min/mg protein in the reisolated microsomal fraction. Considering that equal amounts (protein) of peroxisomes and microsomes were used in the incubation, the above results suggested that a third of the original microsomes sedimented in the peroxisomal fraction. About an equal percentage of labeled PA also sedimented in the peroxisomal fraction.
Therefore, it seems that all of the newly synthesized PA is associated with the microsomes (endoplasmic reticulum vesicles). In Fig. 2, the distribution of lipids in different fractions after correcting for the contamination of peroxisomes by microsomes is shown. It can be seen that almost all of PA remained associated with the microsomes where it is biosynthesized while most of LPA was present in the supernatant fraction (Fig. 2). Biosynthesis and Localization of Lipids Synthesized in Peroxisomes and Microsomes in the Absence of Mg2+ arul BSA-As presented above, Mg2f and BSA stimulated the DHAP acyltransferase reaction probably by preventing the formation of palmitoyl-CoA micelles which are inhibitory to DHAPAT (LaBelle and Hajra, 1972;Declercq et al., 1984). In such a case Mg2+ and BSA may not stimulate the reaction at palmitoyl-CoA concentrations which are below its CMC in the incubation mixture. This was investigated by measuring were added to each tube. After mixing, the mixtures were centrifuged at 7,720 X g for 10 min to sediment peroxisomal fractions (solid bar, H), and the resulting supernatants were centrifuged at 100,000 X g for 90 min to separate microsomes (hatched bur, R) from the soluble fraction (dotted bur, a). The NADPH-cytochrome c reductase activity and the amount of each radioactive lipid present were determined in the aliquots of each fraction. The results presented above were the values corrected for the microsomal contamination of the peroxisomal fractions (see text for details). The values are the average of two experiments (range k 6% of the average values). (Fig. 3). The assay was linear up to 15 min. At this low level of palmitoyl-CoA, there was very little difference between the enzyme activities in the presence or absence of BSA (Fig. 3). This low palmitoyl-CoA concentration was employed to study the biosynthesis and transport of the lipids in the absence of BSA and M P . Fig. 4 shows the results of the biosynthesis and transport of palmitoyl-DHAP, LPA, and localization of PA synthesized in a mixture of peroxisomes and microsomes at low (7 p M ) palmitoyl-CoA concentration and in the absence of BSA and Mg2+. After incubation, the peroxisomes and microsomes in the reaction mixture were separated from each other by differential centrifugation without the addition of EDTA, and the distribution of the radioactive lipids in these fractions and in the supernatant was determined. Under these conditions palmitoyl-DHAP and LPA were present in the supernatant fraction, whereas PA remained associated with the ER vesicles (Fig. 4).
Distribution of Acyl-DHAP and Alkyl-DHAP Biosynthesized in Peroxisomes Using an Acyl-CoA-generating System-Acyl-CoA ligase is present in the peroxisomes (Mannaerts et al., 1982) and the acyl-CoAs generated in peroxisomes are probably physiologically utilized to synthesize acyl-DHAP. Alkyl-DHAP is also biosynthesized in peroxisomes , and this ether lipid should be transported out of peroxisomes to ER to form membrane ether lipids. To study the formation and export of these lipids from peroxisomes under putative physiological conditions, a fatty acyl-CoA-generating system instead of acyl-CoA was used in the presence of DHAP and hexadecanol. The results are shown in Table 11. As seen in this table most (>85%) of the biosynthesized acyl-DHAP and alkyl-DHAP are localized in the soluble fraction and are not associated with peroxisomes. A carrier protein, such as BSA, is not necessary for such export of these keto lipids from peroxisomes where they are biosynthesized (Table 11). The results also show that M$+ does not form insoluble salts with these keto lipids if it remains chelated with excess ATP in the incubation mixtures (Table 11).

DISCUSSION
Results from this cell-free system show that acyl-DHAP synthesized inside the peroxisomes is exported across the membrane and is enzymatically reduced to acyl glycerol-3-P which is transported to the ER where it is acylated to form  PA. Apparently, acyl-DHAP and LPA at low concentrations in the incubation medium (1-16 p~, Table I) are present as water-soluble monomers which could freely diffuse in and out of peroxisomes to the soluble fraction. We have determined the CMCs of palmitoyl-DHAP and LPA under the experimental conditions of peroxisomal lipids biosynthesis and found that the CMCs were 58 p~ for palmitoyl-DHAP and 70 p~ for LPA (see "Appendix"). In contrast, PA at such concentrations is practically insoluble and remains firmly attached to the ER membranes where it is biosynthesized. The enzymatic rate of acylation of LPA to PA is very high, and, consequently, in the presence of microsomes (and NADPH) PA is the major product (Table I and Fig. 1). Although BSA and Mg2+ stimulated peroxisomal DHAPAT, these reagents are not essential for this acyltransferase reaction. At low palmitoyl-CoA concentrations ( 4 0 p~) , there was very little stimulation of the DHAPAT activity by BSA (Fig. 3). As has been reported by different workers (Brandes et al., 1963;LaBelle and Hajra, 1972;Declercq et al., 1984) the stimulation of different acyltransferases by BSA is indirect. BSA binds long chain acyl-CoAs, thus preventing the formation of acyl-CoA micelles which strongly inhibit the acyltransferase (Zahler and Cleland, 1969;LaBelle and Hajra, 1972). The CMC of palmitoyl-CoA has long been a matter of controversy (Zahler et al., 1968;Hsu and Powell, 1975;Powell et al., 1981;Tippett and Neet, 1982), and it varies with different physical parameters such as ionic strength, pH, and temperature of the medium (Powell et al., 1981;Constantinides and Steim, 1985). However, recent careful measurements indicated that at pH 7.0 and low ionic strength the CMC of palmitoyl-CoA is about 40-50 p~ (Smith and Powell, 1986;Constantinides and Steim, 1985), whereas it decreases substantially (8-10 p~) at high ionic strength. Under the conditions of the incubations we used (75 mM Tris-HC1, pH 7.5, 8.3 mM NaF), the CMC of palmitoyl-CoA was determined to be 41 p~ (see "Appendix"). Therefore, the finding that optimum DHAPAT activity is at about 10 p~ of palmitoyl-CoA in the absence of BSA and Mg2+ (Fig. 3) indicates that palmitoyl-CoA monomers, not the micelles, inhibit DHAP acyltransferase. However, as pointed out by Tanford (1980), micelles start to form long before the CMC is reached, and therefore, it is still possible that this acyl transferase is inhibited by palmitoyl-CoA micelles which are present in the incubation mixture at low concentrations of this amphipathic substrate. M e also changes the CMC of acyl-CoAs by forming salt (Constantinides and Steim, 1985), and the observed stimulation of DHAPAT activity by M e in the absence of BSA (Table I and Fig. 1) may also be due to the suppression of palmitoyl-CoA micelle formation in the incubation mixture. Hardeman and van den Bosch (1989) showed that in the presence of BSA all of the acyl-DHAP formed in the rat liver peroxisome is present in the soluble fraction similar to that reported here, but in absence of BSA, most of the biosynthetic acyl-DHAP remained associated with peroxisomes. These authors did not use M$+ in the incubation mixtures to study the biosynthesis of acyl-DHAP. In this respect, these results differ from those presented here. As shown in Fig. 4, in the absence of BSA and M e , palmitoyl-DHAP and LPA formed in peroxisomes were mainly present in the soluble fraction. Palmitoyl-DHAP sedimented with the membrane fraction only in the presence of M$+ (Fig. 1Bl ), but when M e was chelated by EDTA almost all of palmitoyl-DHAP and LPA become soluble even in the absence of BSA (Fig. IC). The reason for the discrepancy between our results and those reported by Hardeman and van den Bosch (1989) is unclear. However, the DHAPAT activity reported by these authors under such conditions (i.e. no BSA, no M$+) is lower (0.9 milliunits/mg protein) (Hardeman and van den Bosch, 1989) than that reported here (3.5 milliunits/mg protein, see Fig. 3) indicating that there may be other differences in the incubation conditions used. Haldar and Lipfert (1990) have also shown that LPA formed from glycerol-3-P in mitochondria is nonsedimentable in the presence of BSA but sediments with mitochondria when BSA is absent. M e was present in this in uitro mitochondrial biosynthesis of LPA (Haldar and Lipfert, 1990) and these authors did not study the effect of removing M e on the partition of LPA in this mitochondrial system. It is not clear, however, why LPA formed in mitochondria has to be exported to ER to form PA when LPA acyltransferase is also present in mitochondria (Haldar et al., 1983). In the present system, because of the absence of LPA acyltransferase in peroxisomes Ballas et al., 1984), these intermediates must be exported to ER t o form PA.
The data presented here demonstrate that acyl-DHAP and LPA formed in peroxisomes are transported by diffusion to ER and converted to PA in the absence of any soluble carrier protein (Fig. 4). In these experiments the acyl-CoA concentrations in the incubation medium was kept low (-7 p~) to prevent inhibition of peroxisomal DHAPAT. Such low acyl-CoA concentration is probably within the physiological range of the liver acyl-CoA concentration which is reported to be 10-20 nmol/g of rat liver (Woldegiorgis et al., 1985). The steady-state concentrations of acyl-DHAP and LPA in rat liver (4 nmol/g and 40 nmol/g, respectively) Hajra, 1984,1989) are also low indicating that these lipids are mainly present as soluble monomers in the cytosol. As mentioned before, the ether analog of acyl-DHAP, i.e. alkyl-DHAP is also biosynthesized in peroxisomes  and is transported to ER to form membrane alkyl-glycerol ether lipids and plasmalogens . In rat liver peroxisomes the activity of the enzyme catalyzing ether bond biosynthesis is very low (Hajra et ul., 1986;Rabert et al., 1986); however, as shown under "Results," the peroxisomally biosynthesized alkyl-DHAP, like acyl-DHAP, is mostly present in the soluble fraction irrespective of the presence or absence of BSA in the incubation medium (Table 11). Hardeman and Van den Bosch (1989) also reported that under the experimental condition they used, alkyl-DHAP was localized in the soluble fraction in the presence of BSA.
Whether or not the in uiuo intracellular transport of these monoacyl or monoalkyl lipids is mediated by a specific carrier protein remains unanswered. Vancura et al. (1991) recently reported that liver mitochondrial lyso-PA formation is stimulated by liver cytosol. They presented preliminary evidence that a soluble 14-kDa protein is the stimulatory factor. We found, however, that peroxisomal biosynthesis of acyl-DHAP under the conditions described in Fig. 4 is not stimulated by the addition of rat liver cytosol.2 It is, of course, possible that a specific liver cytosolic protein, such as fatty acid-binding protein (Glatz and van der Vusse, 1989), may bind acyl-DHAP and LPA thus facilitating their transport between intercellular compartments. However, results presented here demonstrate that such a carrier protein is not essential for the transport of acyl-DHAP and LPA from peroxisomes to microsomes. Almost complete conversion of these peroxisomal lipids to microsomal PA occurs under conditions where these lipids are present as soluble form in a large volume of incubation mixture (no BSA, no M e , Fig. 4) or bound to a soluble protein, i.e. BSA (Fig. lA) or even when present as insoluble salt with Mg2+ in the absence of BSA (Fig. 1B).
Morphological studies showed that peroxisomes and endoplasmic reticulum are closely associated in cells (Novikoff and Novikoff, 1982). Therefore, it seems plausible to assume that in uiuo, with peroxisomes and ER being concentrated in a much smaller cellular volume, such transport takes place without the involvement of a specific carrier protein.