©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synthesis and Intracellular Transport of Aminoglycerophospholipids in Permeabilized Cells of the Yeast, Saccharomyces cerevisiae(*)

(Received for publication, June 15, 1995; and in revised form, September 14, 1995)

Georg Achleitner (1) Dagmar Zweytick (1) Pamela J. Trotter (2) Dennis R. Voelker (2) Günther Daum (1)(§)

From the  (1)Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, A-8010 Graz, Austria and the (2)Lord and Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto Clinical Research Center, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [^3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidylserine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.


INTRODUCTION

In yeast as well as in higher eukaryotes, the endoplasmic reticulum and the inner mitochondrial membrane are generally accepted as the major sites of membrane glycerophospholipid biosynthesis (Bishop and Bell, 1988; Zinser et al., 1991). Other organelles are largely devoid of phospholipid-synthesizing enzymes. This segregation of lipid-synthesizing activity necessitates efficient interorganelle transport to maintain membrane lipid composition, integrity, and function. Several mechanisms of intracellular lipid translocation (for reviews, see Bishop and Bell(1988), van Meer(1989), Trotter and Voelker(1994)) have been proposed including 1) spontaneous and protein-facilitated transport of monomeric phospholipids through the cytosol; 2) vesicle budding, translocation, and fusion; 3) regulated organelle juxtaposition and/or contact. Currently there is sufficient evidence to implicate each of the proposed mechanisms depending upon the donor and acceptor membrane and the lipid transferred.

The topological segregation of yeast enzymes involved in aminoglycerophospholipid synthesis: phosphatidylserine synthase in the endoplasmic reticulum or closely related membranes (Zinser et al., 1991; Gaigg et al., 1995); phosphatidylserine decarboxylase 1 in mitochondria and phosphatidylserine decarboxylase 2 in the Golgi and vacuole (Trotter et al., 1995); and phosphatidylethanolamine methyltransferases in the endoplasmic reticulum (Kuchler et al., 1986), enables the sequential metabolism of phosphatidylserine phosphatidylethanolamine phosphatidylcholine to be used to follow interorganelle lipid transport. This technique has been applied to intact and permeabilized mammalian cells, as well as to isolated organelles (for a review, see Trotter and Voelker(1994)). The rate of phosphatidylserine transfer to mitochondria was greatly reduced by ATP depletion in intact cells (Voelker, 1985) and stimulated by ATP addition in permeabilized cells (Voelker, 1989b, 1990, 1993). Interorganelle translocation of phosphatidylserine was shown to be largely independent of soluble cytosolic proteins in permeabilized mammalian cells (Voelker, 1989b) and in a cell free system (Voelker, 1989a). Membrane collision and/or vesicle flux have been suggested as possible mechanisms of this transport process. Membrane contact of mitochondria with a phospholipid-synthesizing microsomal fraction (Vance, 1990; Ardail et al., 1993; Gaigg et al., 1995) is probably the basis of lipid transport between these two organelles. Work with disrupted permeabilized mammalian cells provided evidence that structural elements coupling a donor membrane compartment to the mitochondria were involved in phosphatidylserine transport (Voelker, 1993).

The use of [^3H]serine metabolism to [^3H]phosphatidylethanolamine as a specific measure of phosphatidylserine transport to the mitochondria has come under closer scrutiny as a result of the recent finding that there are two phosphatidylserine decarboxylases, one in the mitochondria (PSD1) (Trotter et al., 1993; Clancey et al., 1993) and the other in the Golgi apparatus and the vacuole (PSD2) (Trotter and Voelker, 1995; Trotter et al., 1995). The availability of well defined psd1 and psd2 mutants (Trotter et al., 1993, 1995; Trotter and Voelker, 1995) now makes it possible to evaluate which transport pathways are being followed in the metabolism of nascent phosphatidylserine.

In this report we describe the optimization of the permeabilized yeast cell technique in order to 1) define the requirements for aminoglycerophospholipid synthesis and transport, 2) discriminate between nascent phosphatidylserine transport to the mitochondria and Golgi plus vacuoles, and 3) define the energetic requirements for phosphatidylserine transport to, and phosphatidylethanolamine transport from, the mitochondria. We present evidence that the PSD1 gene product catalyzes synthesis of the vast majority of cellular phosphatidylethanolamine via decarboxylation in permeabilized cells.


EXPERIMENTAL PROCEDURES

Strains and Culture Conditions

The wild-type yeast strains Saccharomyces cerevisiae X-2180 (aSUC2 mal gal2 CUP1) and S. cerevisiae 422 (alphatrp1-289 leu2-3, 112 his7 lys2) (kindly provided by Dr. R. Sclafani, Denver) and the phosphatidylserine decarboxylase-deficient yeast strains PTY13 (alphatrp1 leu2 his lys2 psd1-Delta1::TRP1), PTY22 (atrp1 leu2 his ade1 lys2 psd2), and PTY18 (atrp1 leu2 his ade1 psd1-Delta1::TRP1 psd2) were grown on YPD medium (1% yeast extract, 2% peptone, 3% glucose) under aerobic conditions at 30 °C to an A of 2-4. The PTY18 strain was additionally supplemented with 2 mM ethanolamine.

Preparation of Spheroplasts, Permeabilized Yeast Cells, and Yeast Subcellular Fractions

Yeast spheroplasts were prepared by the method of Daum et al.(1982) with the modification that YPD (0.5% glucose) was present during treatment of cells with zymolyase. Permeabilized yeast cells were prepared essentially as described by Baker et al.(1988) with minor modifications. In brief, spheroplasts regenerated for 20 min at 30 °C in the presence of 0.75% yeast extract, 1.5% peptone, 1% glucose, and 0.7 M sorbitol were washed with lysis buffer consisting of 0.4 M sorbitol, 20 mM HEPES, pH 6.8, 0.15 M potassium acetate, 2 mM magnesium acetate, and 0.5 mM EGTA. Then, spheroplasts were suspended in the lysis buffer at a concentration of approximately 0.5 g wet weight per ml. The suspension was divided into 0.2-0.3-ml portions, filled in Eppendorf tubes, and frozen over liquid nitrogen. Freezing was complete within 15 min. At this stage, cells were stored at -75 °C, and used within the next 3 months. For thawing, the Eppendorf tubes with the cell suspension were placed on ice, and this resulted in an optimal degree of cell permeabilization, but left organelles largely intact. During all following steps care was taken to minimize mechanical shear forces. The degree of permeabilization was evaluated by microscopic inspection, marker enzyme measurements, and Western blotting. For the latter procedure, 20 µl of the suspension of permeabilized cells were diluted with 0.98 ml of 1.2 M sorbitol and centrifuged for 5 min at 4 °C at 6,500 rpm in an Eppendorf tabletop centrifuge. The supernatant, which should contain only the cytosol, and the pellet, which should contain permeabilized cells, were tested with antibodies against cytosolic glyceraldehyde-3-phosphate dehydrogenase, mitochondrial porin, 40-kDa microsomal protein, Kex2 protease, and carboxypeptidase Y. The cytosolic marker enzyme glyceraldehyde-3-phosphate dehydrogenase was measured by the the method of Byers(1982), and glucose-6-phosphate dehydrogenase by the method of Deutsch(1983). In standard preparations, the degree of permeabilization was found to be approximately 85-95% as judged from the remaining cytosol in the cellular pellet. Organelles probed with antibodies mentioned above were more or less completely recovered in permeabilized cells in the pellet. Intactness of mitochondria was tested using an antibody against cytochrome b(2), an intermembrane space protein (Daum et al., 1982), and found to be unaffected by the cell permeabilization procedure.

Cell homogenates were prepared by suspending permeabilized yeast cells in 0.6 M mannitol, 10 mM Tris-Cl, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (0.2 mg of protein per ml), and disrupting with 10 strokes in a Dounce homogenizer. Mitochondria and microsomal fractions were prepared as described elsewhere (Daum et al., 1982; Zinser et al., 1991).

Synthesis of Aminoglycerophospholipids in Permeabilized Yeast Cells

A typical assay mixture for 8 time points to be taken contained 2 mg of cellular protein (permeabilized cells) in a total volume of 0.9 ml. The assay mixture consisted of 90 µl of 6 mM MnCl(2), 90 µl of water, 195 µl of 1.2 M sorbitol, 400 µl of assay buffer (0.6 M mannitol, 50 mM TrisCl, pH 8.0), and 50 µl of permeabilized yeast cells (0.5 g wet weight per ml). Permeabilized cells were added last to avoid an osmotic shock. The reaction was started by the addition of 45 µl of 2 mM unlabeled serine and 30 µl of [^3H]serine (specific activity 15-40 Ci/mmol; 30 µCi in 0.9-ml assay volume). The incubation temperature was 30 °C, and samples of 0.1 ml were taken at the time points indicated. Under these conditions, synthesis of phosphatidylserine occurred.

After 15 min of incubation, EDTA (stock solution: 0.1 M in 0.6 M mannitol, 50 mM TrisCl, pH 8.0) was added to a final concentration of 4 mM. EDTA stopped the synthesis of phosphatidylserine and stimulated the synthesis of phosphatidylethanolamine. Samples of 0.1 ml plus the aliquot volume of added EDTA were taken in this second phase.

After 15 min of further incubation, MgCl(2) (final concentration 8 mM) and S-adenosylmethionine (AdoMet(^1); final concentration 0.23 mM) were added. A stock solution of 10 mM AdoMet was prepared by dissolving an appropriate amount of AdoMet in 0.6 M mannitol, 50 mM Tris-Cl and adjusting to pH 8.0 with Tris base. The AdoMet stock solution was freshly prepared for every experiment. MgCl(2) was prepared as a 0.1 M stock solution in 0.6 M mannitol, 50 mM Tris-Cl, pH 7.5. In the presence of MgCl(2) and AdoMet, phosphatidylcholine was synthesized. Samples of 0.1 ml plus the aliquot volumes of added EDTA, MgCl(2), and AdoMet were taken in the third phase of the incubation.

MgATP or MnATP, an ATP-regenerating system (10 units of creatine phosphokinase per mg of protein and 5 mM creatine phosphate), N-ethylmaleimide, adriamycin, apyrase, oligomycin, and azide were added at final concentrations as indicated in Table 2. Permeabilized cells were preincubated with MgATP and inhibitors for 5 min on ice and with apyrase for 30 min at 30 °C. In order to remove cytosol, permeabilized cells were washed twice with 0.2 ml of lysis buffer and reisolated by centrifugation at 1,000 times g for 30 s in an Eppendorf tabletop centrifuge. The same washing procedure was employed to remove apyrase from permeabilized cells. Subsequently, cells were carefully suspended in the respective incubation buffer.



When radiolabeled phospholipids were localized in subcellular fractions, 3.5 mg of permeabilized cells were incubated in 1.8 ml of the assay mixture as described above. After 20, 40, and 120 min of incubation under appropriate conditions, 0.4-ml samples were taken and chilled, and 5 mM azide and fluoride and 8 mM EDTA and hydroxylamine (final concentration, each) were added. Mitochondria and 40,000 times g microsomes (2 mg of protein, each) were added as carrier, and the respective fractions were isolated by standard procedures (Daum et al., 1982; Zinser et al., 1991). Azide, fluoride, EDTA, and hydroxylamine (concentrations see above) were present throughout the isolation procedure. The purity of fractions was essentially the same as described by Zinser et al. (1991).

Extraction and Analysis of Phospholipids

Aliquots (0.1-0.116 ml) of the incubation mixture (see above) were extracted with 4 ml of chloroform/methanol (2:1, v/v) for 1 h at room temperature with repeated vortexing. Prior to the extraction carrier, phospholipids (approximately 50-100 µg of soybean phospholipids, Epicuron) were added. The organic phase was washed once with 2 ml of 0.034% MgCl(2), once with 2 ml of 2 M KCl/methanol (4:1, v/v), and once with 2 ml of methanol/water/chloroform (48:47:3, v/v). 2 ml of the resulting organic phase were withdrawn and taken to dryness under a stream of nitrogen. Lipids were dissolved in 30 µl of chloroform/methanol (2:1, v/v) and subjected to thin layer chromatography on Silica Gel 60 (Merck, Darmstadt, Germany) or Silica Gel H (Analtech, Newark, DE). Chloroform, methanol, 25% ammonia (50:25:6, v/v) or chloroform, methanol, 2-propyl alcohol, triethylamine, 0.25% KCl in water (90:27:75:54:18, v/v) were used as developing solvent. Spots were visualized in iodine vapor or by spraying the thin layer plates with 0.1% aqueous 8-anilino-1-naphthalenesulfonic acid and exposure to UV light. Individual phospholipids were identified with authentic standards, scraped from thin layer plates, and counted in 8 ml of Safety Mixture (Baker) plus 5% water or 4.5 ml of Scintisafe (Fisher) plus 0.5 ml water.

Miscellaneous Methods

Proteins were quantitated by the method of Lowry et al.(1951), or using the BCA protein assay system (Pierce). SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and Western blotting (Haid and Suissa, 1983) were carried out by published procedures. Immunoreactive proteins on nitrocellulose sheets were detected by enzyme-linked immunosorbent assay techniques with secondary antibodies coupled to peroxidase or alkaline phosphatase following the manufacturer's instructions. Stained bands were quantified by densitometric scanning using a Shimadzu CS 930 chromatoscanner.

Cellular ATP was quantified using the firefly luciferase bioluminescence assay as described by Lundin(1982).

Reproducibility of Experiments

Data shown in the figures are from one representative of at least three independent experiments. Results of these experiments were essentially the same, but absolute values varied slightly due to variance in the uptake of [^3H]serine, the degree of permeabilization of spheroplasts, and the amount of cells used. Permeabilized yeast cells were used for assays only when the degree of permeabilization was higher than 80%.


RESULTS AND DISCUSSION

Yeast Cells Can Be Efficiently Permeabilized for Studies of Synthesis and Intracellular Transport of Phospholipids

In contrast to mammalian cells, permeabilized yeast cells have not been as widely used to study problems of intracellular transport of macromolecules. Despite a low degree of permeabilization (approximately 50%), the yeast system yielded reliable results for the study of the protein secretory pathway (Baker et al., 1988), because externally supplied macromolecules were not able to enter intact spheroplasts and were functional in permeabilized cells only. By comparison, a low degree of permeabilization would make results presented in this study difficult to interpret, because the low molecular weight precursor of aminoglycerophospholipids, [^3H]serine, can easily cross the plasma membrane of intact spheroplasts. For this reason we optimized the permeabilization procedure of yeast cells for our purposes. In brief, rates of freezing and thawing were found to strongly influence the quality and the stability of permeabilized cells. As can be seen from Table 1, the procedure outlined under ``Experimental Procedures'' yielded a high degree of permeabilization with minimal disruption of subcellular structures (organelles). At least 90% of the soluble enzyme glyceraldehyde-3-phosphate dehydrogenase was released from the cells as determined by either immunoblot analysis or enzyme activity measurement. Similar results were obtained by the activity measurement of glucose-6-phosphate dehydrogenase. Measurement of proteins associated with the mitochondrial outer membrane (porin) or the intermembrane space (cytochrome b(2)) detected by immunoblotting demonstrated that mitochondria were at least 90% retained in the permeabilized cell pellet. These latter results also clearly demonstrate that the structural integrity of the mitochondria were not compromised by permeabilization. Additives to the permeabilized cell preparation, e.g. EDTA or AdoMet (see below), do not gain access to organelles of intact spheroplasts. The effect of EDTA and AdoMet were apparent only in permeabilized cells and served as additional independent indicators for the degree of permeabilization.



Permeabilized Yeast Synthesize Phosphatidylserine and Metabolize It to Phosphatidylethanolamine and Phosphatidylcholine

The synthesis of phosphatidylserine and its subsequent metabolism to phosphatidylethanolamine and phosphatidylcholine can be followed readily using [^3H]serine as a precursor. Although CDP-diacylglycerol is also required for phosphatidylserine synthesis, we found that sufficient levels of this liponucleotide were present in the permeabilized yeast cells to support the first step of aminoglycerophospholipid synthesis for at least 120 min. This situation is advantageous because it avoids the addition of exogenous CDP-diacylglycerol which has some detergent properties and can compromise organelle integrity.

The synthesis of phosphatidylserine depends on the presence of divalent cations, especially of Mn, in the incubation mixture (Bae-Lee and Carman, 1984; Sperka-Gottlieb et al., 1990). The subsequent step in the biosynthetic pathway of aminoglycerophospholipids, the decarboxylation of phosphatidylserine leading to phosphatidylethanolamine, is inhibited by divalent cations and stimulated in the presence of EDTA (Lamping et al., 1991). Finally, conversion of phosphatidylethanolamine to phosphatidylcholine needs the addition of S-adenosylmethionine (AdoMet) and the presence of Mg (Kodaki and Yamashita, 1989). The different optima for each step in aminoglycerophospholipid synthesis necessitated the design of a three-step assay, which fulfills all the above-mentioned requirements.

In a standard experiment shown in Fig. 1(details given under ``Experimental Procedures''), permeabilized yeast cells were first incubated for 15 min with [^3H]serine in the presence of Mn to form phosphatidylserine. Subsequently, EDTA was added, and the formation of phosphatidylethanolamine was followed for another 15 min. As can be seen from Fig. 1, practically no phosphatidylethanolamine was formed during the first phase of the incubation, but the addition of EDTA stopped the synthesis of phosphatidylserine and stimulated its decarboxylation to phosphatidylethanolamine. Without addition of EDTA, no phosphatidylethanolamine was formed (data not shown).


Figure 1: Aminoglycerophospholipid biosynthesis in permeabilized yeast cells. Permeabilized cells of the wild-type strain, S. cerevisiae X-2180, were prepared and incubated with [^3H]serine as described. During the first 15 min, the incubation mixture contained 0.6 mM Mn. During the second 15 min, EDTA was included at a final concentration of 4 mM. After 30 min of incubation, Mg (8 mM final concentration) and AdoMet (SAM) (0.23 mM final concentration) were added. Aliquots of the incubation mixture were removed at the time points indicated, and the lipids were extracted and analyzed by thin layer chromatography. Values for phosphatidylserine (A), phosphatidylethanolamine (B), and phosphatidylcholine (C) are expressed as disintegrations/min/µg of cellular protein. Data shown in the figure are from a representative of three independent experiments.



Finally, AdoMet and Mg were added, and the formation of phosphatidylcholine was followed for another 70 min (see Fig. 1). In control experiments carried out without AdoMet and Mg, no phosphatidylcholine was synthesized (data not shown). During this phase of the assay, incorporation of [^3H]serine into phosphatidylserine was observed again, because phosphatidylserine synthase was reactivated in the presence of divalent cations. Phosphatidylethanolamine formation also continued under these conditions indicating that Mg inhibition may only be relevant in assays using solubilized enzyme. Radioactivity was not only found in phosphatidylcholine, but also in phosphatidyldimethylethanolamine and lysophosphatidylethanolamine (5 and 10% of labeled phospholipids, respectively). For the sake of clarity, only values of phosphatidylcholine, the fully methylated end product in the sequence of biosynthetic steps of aminoglycerophospholipids, are shown (see also Fig. 1).

In Permeabilized Yeast the Majority of Nascent Phosphatidylserine That Is Decarboxylated Is Transported to the Mitochondria

The occurrence of phosphatidylserine decarboxylase activity in two different compartments of the yeast (Trotter et al., 1995; Trotter and Voelker, 1995) raises the possibility that phosphatidylserine destined to be converted to phosphatidylethanolamine could be translocated to the Golgi or the vacuole in addition to being imported into mitochondria. In vivo, the activity derived from the extramitochondrial gene product is sufficient for a balanced phospholipid metabolism and cellular growth (Trotter et al., 1993). In principal, the total amount of phosphatidylethanolamine required for yeast growth can be synthesized without the participation of the mitochondrial phosphatidylserine decarboxylase. In order to clarify which pathway contributes to phosphatidylethanolamine synthesis in permeabilized cells, we conducted experiments using yeast strains with psd1 (mitochondrial) and psd2 (extramitochondrial) mutations. Permeabilized cells containing the psd1 null allele (Fig. 2B) formed [^3H]phosphatidylethanolamine at less than 5% of the wild-type control (Fig. 2A), and only trace amounts of phosphatidylcholine were formed. In contrast, permeabilized cells with the psd2 mutation (Fig. 2C) exhibited significant synthesis of phosphatidylethanolamine (via decarboxylation) and phosphatidylcholine. The strain with both the psd1 and psd2 mutations, which completely lacks detectable phosphatidylserine decarboxylase activity, produced radioactive phosphatidylethanolamine and phosphatidylcholine at only background levels (Fig. 2D). These data demonstrate that the majority of phosphatidylethanolamine synthesized in permeabilized yeast cells is formed primarily by the PSD1 gene product. Thus, the appearance of phosphatidylethanolamine in these experiments likely indicates the transport of phosphatidylserine to the inner mitochondrial membrane, and not to the location of the PSD2 gene product.


Figure 2: Phosphatidylserine decarboxylase 1 produces the majority of phosphatidylethanolamine formed in permeabilized yeast. Permeabilized cells of the yeast strains S. cerevisiae 422 (wild-type) (A), PTY13 (psd1-Delta1::TRP1) (B) with a defect in the mitochondrial phosphatidylserine decarboxylase, PTY22 (psd2) (C) defective in a second extramitochondrial phosphatidylserine decarboxylase activity, and PTY18 (psd1-Delta1::TRP1Deltapsd2) (D), a double mutant lacking both phosphatidylserine decarboxylase activities, were prepared and incubated with [^3H]serine as described under ``Experimental Procedures'' and in the legend to Fig. 1. Data shown in the figure are from a representative of three independent experiments. , phosphatidylserine; bullet, phosphatidylethanolamine; , phosphatidylcholine. SAM, AdoMet.



Nascent Phosphatidylserine Import into Mitochondria of Permeabilized Yeast Is ATP-independent

From the findings presented above we conclude that formation of phosphatidylethanolamine from radiolabeled phosphatidylserine is a valid measure of the import of phosphatidylserine into mitochondria. In permeabilized mammalian cells, the translocation of phosphatidylserine from its site of synthesis to its site of conversion to phosphatidylethanolamine was shown to be ATP-dependent (Voelker, 1989b, 1990, 1993) and inhibited by adriamycin (Voelker, 1991). Transport between isolated organelles or within permeabilized cells did not require cytosolic components (Voelker, 1989a, 1989b, 1990, 1993). Similar parameters were now examined with permeabilized yeast cells (Table 2). Removal of cytosol from permeabilized cells by repeated washes was shown to be without effect on the synthesis, transport, and decarboxylation of phosphatidylserine. Addition of MgATP to the assay mixture did not have a stimulatory effect on the formation of phosphatidylethanolamine. This latter result could be due to the high levels of ATP present in the spheroplasts prior to permeabilization. However, preincubation of permeabilized cells with oligomycin and apyrase which deplete cellular ATP practically to negligible levels (as measured by the luciferin-luciferase assay) was also ineffective at altering phosphatidylserine transport. The discrepancies between the mammalian and the yeast system may be attributable to differences in phosphatidylserine synthesis between the two types of cells. Yeast phosphatidylserine synthesis is a CDP-diacylglyceroldependent reaction (see Paltauf et al.(1992)), whereas the mammalian enzyme catalyzes base exchange (see Bishop and Bell, 1988). Both enzyme systems are microsomally located, but may result in different physical topology of nascent phosphatidylserine. As a consequence, the accessibility of phosphatidylserine to the lipid translocation machinery may be different, which could in turn cause different requirements for intracellular transport. In the mammalian system, ATP is required for both translocation and synthesis of phosphatidylserine (Voelker, 1990, 1993); phosphatidylserine synthesis is coupled to the action of the Ca-sequestering ATPase of the endoplasmic reticulum. Yeast phosphatidylserine synthase is not Ca-dependent. The enzymatic reaction is energy-dependent due to the requirement of CDP-diacylglycerol as a co-substrate, but is not directly affected by ATP.

Permeabilized yeast cells preincubated with adriamycin exhibited a higher rate of phosphatidylserine biosynthesis than untreated cells (data not shown). However, in contrast to mammalian cells, adriamycin did not affect the import of phosphatidylserine into mitochondria of permeabilized yeast cells (see Table 2). This observation is consistent with previous findings by Simbeni et al.(1993) obtained in vitro with isolated yeast mitochondria. Adriamycin binds with high affinity to cardiolipin (Goormaghtigh et al., 1984), a lipid that is present at high concentrations in contact sites between the outer and the inner mitochondrial membrane (Ardail et al., 1990; Simbeni et al., 1991). The interaction of adriamycin with cardiolipin is one piece of evidence implicating zones of mitochondrial membrane contact as sites of protein import in yeast (Eilers et al., 1989) and phospholipid import in mammalian cells. The differential effects of adriamycin upon yeast and mammalian lipid transport remain unclear. In mammalian cells, it is possible that adriamycin affects an earlier step than contact site transit of phosphatidylserine, which may not be identical with that of the yeast system.

Contact between phospholipid-synthesizing membranes and mitochondria has been suggested as a possible prerequisite for the import of phospholipids into mitochondria of mammalian cells (Voelker 1989a, 1990, 1993; Vance, 1991; Ardail et al., 1991) and yeast (Gaigg et al., 1995). In permeabilized mammalian cells that are disrupted by shearing, transport of nascent phosphatidylserine appears to be restricted by specific structural association between the mitochondria and the membrane compartment synthesizing the lipid (Voelker, 1993). As found for mammalian cells, disruption of permeabilized yeast by homogenization yields cell preparations that remain competent to synthesize, transport, and metabolize phosphatidylserine (see Table 2).

Phosphatidylethanolamine Export from Mitochondria to the Locus of the Methyltransferases Requires ATP

Phosphatidylethanolamine formed in mitochondria by the action of phosphatidylserine decarboxylase can be further metabolized to phosphatidylcholine by the nonmitochondrial AdoMet-dependent phosphatidylethanolamine-N-methyltransferase and phospholipid-N-methyltransferase (Kuchler et al., 1986; Gaigg et al., 1995). Therefore, export of phosphatidylethanolamine from mitochondria is required to deliver the substrate to the methyltransferases.

Preliminary characterization of phosphatidylethanolamine export from the mitochondria as monitored by phosphatidylcholine formation revealed it did not require cytosol and was inhibited by the addition of apyrase plus oligomycin. Additional experiments, however, demonstrated that methyltransferase activity was also markedly inhibited by apyrase plus oligomycin treatment. This latter finding made phosphatidylcholine formation an unreliable indicator for phosphatidylethanolamine export from the mitochondria in the presence of apyrase and oligomycin. In order to critically test the role of ATP in phosphatidylethanolamine export from mitochondria, we carried out subcellular fractionation of permeabilized yeast cells after each stage of incubation, i.e. after 20, 40, and 120 min of incubation under the optimized conditions for phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine synthesis. Appearance of phosphatidylethanolamine in microsomes served as an indicator of its export from mitochondria. In the presence of apyrase (Fig. 3A), phosphatidylethanolamine transport from mitochondria to microsomes was dramatically decreased. As a consequence, conversion of phosphatidylethanolamine to phosphatidylcholine could not occur (Fig. 3B). In control assays without apyrase, phosphatidylethanolamine reached the microsomal fractions and was, in part, further converted to phosphatidylcholine. This result confirmed the idea that export of phosphatidylethanolamine from mitochondria is an energy-dependent step in this part of the pathway leading to phosphatidylcholine thus confirming previous findings with intact yeast cells (Daum et al., 1986; Gnamusch et al., 1992). Data shown in Fig. 3, A and B, also demonstrate that phosphatidylcholine formed in microsomes of untreated cells can be readily transported back to mitochondria.


Figure 3: Phosphatidylethanolamine export from mitochondria requires ATP but not ongoing methyltransferase activity. Permeabilized yeast cells were labeled with [^3H]serine as described under ``Experimental Procedures.'' After 40 min (time point of AdoMet addition) and 120 min, cells were harvested and fractionated. Mitochondria and the endoplasmic reticulum (microsomes; 40,000 times g pellet) were analyzed for specific radioactivity in aminoglycerophospholipids. A, C, and E, ratio of disintegrations/min in phosphatidylethanolamine (PE) to phosphatidylserine (PS). In a typical experiment, control mitochondria contain approximately 20,000 dpm, and microsomes 1,000 dpm in phosphatidylethanolamine. B, D, and F, % disintegrations/min in phosphatidylcholine (PC) with control mitochondria and control microsomes, respectively, set at 100%. In a typical experiment, control mitochondria contain approximately 2,000 dpm, and microsomes 300 dpm in phosphatidylcholine. A and B, assays were carried out in the absence (control) or presence of apyrase and oligomycin (ATP-depleted). C and D, permeabilized cells were de-energized and washed as described under ``Experimental Procedures.'' An ATP-regenerating system (ATP reg.) was added to the control, whereas apyrase was present during prolonged incubation in the second assay (+ Apyrase). E and F, assays were carried out in the presence (control) or in the absence (without SAM) of S-adenosylmethionine.



When de-energized permeabilized yeast cells were re-energized by washing and addition of ATP, export of phosphatidylethanolamine from mitochondria and formation of phosphatidylcholine recovered (Fig. 3, C and D). Ongoing methylation of phosphatidylethanolamine was not the limiting factor of the translocation step, because in the absence of AdoMet phosphatidylethanolamine reached the endoplasmic reticulum (Fig. 3E), although further conversion to phosphatidylcholine could not occur (Fig. 3F). Thus, the methylation of phosphatidylethanolamine did not exert a feedback control on its export from mitochondria.


CONCLUSIONS

While the precise mechanism of phospholipid translocation between organelles remains uncertain, circumstantial evidence continues to accrue in favor of membrane contact as an important prerequisite for this process. Membranes with a high capacity to synthesize phospholipids were found to be associated with mitochondria of mammalian (Vance, 1990, 1991; Ardail et al., 1991, 1993) as well as of yeast cells (Gaigg et al., 1995). In addition, Voelker(1993) demonstrated that transport and decarboxylation of phosphatidylserine in disrupted mammalian cells is restricted to those mitochondria associated with the phosphatidylserine-synthesizing membrane. The observation that homogenates obtained from permeabilized spheroplasts readily translocated phosphatidylserine and phosphatidylethanolamine between endoplasmic reticulum-related membranes and mitochondria indicates the process is very efficient in a cell-free system. One explanation of these results is that stable organelle contact might be the basis of lipid translocation. Alternatively, vesicle transport along structural corridors that tether different organelles can also be envisaged as a possible mechanism of phospholipid translocation. The role of cytosolic lipid transfer proteins (Wirtz, 1991), which catalyze lipid migration in vitro, as mediators of lipid transport in living cells still remains obscure despite extensive genetic and molecular biological studies (Bankaitis et al., 1989; 1990; Cleves et al., 1991a; 1991b) with the yeast phosphatidylinositol transfer protein. Another cytosolic yeast lipid transfer protein, which catalyzes phosphatidylserine and phosphatidylethanolamine transfer in vitro (Lafer et al., 1991), was originally regarded as a candidate to facilitate the transport of phosphatidylserine into, and of phosphatidylethanolamine out of, mitochondria. The fact that removal of cytosol from permeabilized yeast cells affects neither phosphatidylserine nor phosphatidylethanolamine translocation between the endoplasmic reticulum and mitochondria argues against such a function of the phosphatidylserine transfer protein. This result is in agreement with the observation that translocation of phosphatidylserine between the endoplasmic reticulum and mitochondria of mammalian cells in vitro is not stimulated by cytosolic proteins (Voelker, 1989a).

Finally, it should be considered that all membranes of a cell, which are not able to synthesize their own lipids, rely on the supply of lipids from synthesizing organelles. Most membranes of eukaryotic organelles contain the whole set of phospholipids, although at different amounts and proportions. If lipid-synthesizing membranes, e.g. the endoplasmic reticulum, associate with other cellular membranes in order to translocate lipids, contact between organelles could predetermine the subcellular distribution of lipids. In this case, physical factors governing membrane contact could regulate the assembly of lipids into membranes.


FOOTNOTES

*
This work was financially supported by Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich Project S-5811 (to G. D.) and National Institutes of Health Grants GM 32453 (to D. R. V.) and GM 16701 (to P. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut für Biochemie und Lebensmittelchemie, Technische Universität Graz, Petersgasse 12/2, A-8010 Graz, Austria. Tel.: 43-316-873-6462; Fax: 43-316-873-6952; daum@ftug01.dnet.tu-graz.ac.at.

(^1)
The abbreviation used is: AdoMet, S-adenosylmethionine


ACKNOWLEDGEMENTS

We thank F. Paltauf for helpful discussions during the preparation of the manuscript. Antiserum against carboxypeptidase Y was kindly provided by D. Wolf, Stuttgart, Germany.


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