(Received for publication, June 15, 1995; and in revised form, September 14, 1995)
From the
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
[H]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.
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 [H]serine metabolism to
[
H]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.
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).
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 (final
concentration 8 mM) and S-adenosylmethionine
(AdoMet(
); 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
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
and AdoMet, phosphatidylcholine was synthesized. Samples of 0.1
ml plus the aliquot volumes of added EDTA, MgCl
, 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 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 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).
Cellular ATP was quantified using the firefly luciferase bioluminescence assay as described by Lundin(1982).
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 [H]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
[H]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
[
H]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).
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 (psd1-1::TRP1) (B) with a defect in the
mitochondrial phosphatidylserine decarboxylase, PTY22 (psd2) (C) defective in a second extramitochondrial
phosphatidylserine decarboxylase activity, and PTY18 (psd1-
1::TRP1
psd2) (D), a double mutant
lacking both phosphatidylserine decarboxylase activities, were prepared
and incubated with [
H]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;
,
phosphatidylethanolamine;
, phosphatidylcholine. SAM,
AdoMet.
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).
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
[H]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
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.
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.