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INTRODUCTION |
The biochemical, molecular, and genetic details of the
interorganelle transport of phospholipids are poorly understood. One approach to studying this problem utilizes the organelle-specific metabolism of the aminoglycerophospholipids, phosphatidylserine (PtdSer),1
phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho) outlined in Fig. 1 (1). In yeast, PtdSer is synthesized in the
endoplasmic reticulum and related membranes (2). The newly formed
PtdSer is subsequently decarboxylated to form PtdEtn in the
mitochondria, by PtdSer decarboxylase 1 (Psd1p) (3); and in the
Golgi/vacuole, by PtdSer decarboxylase 2 (Psd2p) (4). The PtdEtn
resulting from the action of the decarboxylases is exported from the
mitochondria and Golgi/vacuole to the endoplasmic reticulum, where it
is methylated to form PtdCho (3, 5). This sequential and subcellular
location-specific metabolism of the aminoglycerophospholipids provides
a rapid and convenient method for assessing lipid transport in intact
cells, permeabilized cells, and isolated organelles (1). This
intracellular site-specific metabolism has also provided a means to
develop genetic screens for new yeast strains defective in the
interorganelle transport of phospholipids (6, 7). By using yeast
strains with selective genetic defects in expression of either Psd1p
(psd1
mutants) or Psd2p (psd2
mutants), it
is possible to study lipid trafficking events to either the
Golgi/vacuole or the mitochondria, respectively, using both biochemical
and genetic approaches (6-8). Currently, two mutant strains have been
identified using a genetic approach, and these strains identify lesions
in a phosphatidylinositol 4-kinase (Stt4p) (6), and a
phosphatidylinositol transfer protein (PstB2p) (7).
To complement the genetic approach and studies performed with intact
cells, it is necessary to develop an in vitro assay system amenable to the addition and deletion of specific macromolecules to
enable further biochemical and mechanistic dissection of the transport
processes. Such a system will also allow the mixing of different donor
and acceptor populations of organelles that localizes lesions
topologically. Previous work by Achleitner et al. (8)
described conditions for transport of nascent PtdSer from the ER to
Psd1p in the mitochondria in permeabilized yeast cells. These preceding
studies did not reveal conditions for transport of PtdSer from the ER
to the locus of Psd2p in the Golgi/vacuole. The objectives of our
current studies were to 1) define conditions for reconstituting PtdSer
transport to Psd2p in permeabilized cells; 2) establish conditions for
defining separable donor and acceptor compartments for PtdSer synthesis
and decarboxylation; 3) identify the role of nucleotides and divalent
cations in the process; and 4) apply the reconstituted system to
defining the site of action of PstB2p. In this report, we now describe
the characteristics of PtdSer transport from the ER to the Golgi of permeabilized cells.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All chemicals, including amino acids for yeast
media, were purchased from Sigma or Fisher. Other components for yeast
growth media were purchased from Difco. Phospholipids were obtained
from Avanti Polar Lipids. Thin layer silica gel H plates were from Analtech Corp. The radioisotopes [3-3H]serine and
[1-14C]serine were purchased from Amersham Pharmacia
Biotech and ICN, respectively. The lipid
1-acyl,2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl)aminocaproyl-Ptd-[1'-14C]serine
was synthesized using Escherichia coli PtdSer synthase (9).
Protein determination reagents were obtained from either Pierce or
Bio-Rad. Mouse monoclonal antibody against the 100-kDa subunit of yeast
vacuolar H+-ATPase was purchased from Molecular Probes,
Inc. (Eugene, OR). Other reagents used for ligand blotting were from
Bio-Rad and Sigma.
Strains and Culture Conditions--
The Psd1p-deficient yeast
strains PTY40 (MAT
ura3 his3 trp1 met14
psd1
-1::TRP1) and WWY66 (MAT
lys2 trp1 ura3 his3 leu2 suc2
psd1
-1::TRP1 pstB2::HIS3) were
cultured in YPDAUE (standard YPD medium supplemented with additional
adenine, uracil, and ethanolamine) (6) medium under aerobic conditions
at 30 °C to an A600 between 1 and 2.
Preparation of Permeabilized Yeast Cells--
Permeabilized
cells were prepared in lysis buffer (20 mM HEPES, pH 6.8, 0.15 M potassium acetate, 2 mM magnesium
acetate, and 0.5 mM EDTA) following the method described by
Achleitner et al. (8). In brief, yeast spheroplasts were
prepared by treating cells with dithiothreitol under alkaline
conditions, followed by zymolyase treatment in the presence of low
glucose (0.5%) YPD (10). The resulting spheroplasts were 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 before they were
washed and then resuspended in lysis buffer at a concentration of
0.5 g, wet weight/ml. The cell suspensions were divided into
0.2-0.3-ml aliquots and frozen over liquid nitrogen vapor for 15 min.
The frozen cells could be stored at
80 °C for at least 3 months.
PtdSer Transport Assays--
[3H]serine was
incorporated into the permeabilized cells in a reaction mixture
containing 8 mM HEPES, 22 mM Tris-HCl, pH 8, 0.6 mM MnCl2, 0.8 mM magnesium
acetate, 60 mM potassium acetate, 0.1 M KCl,
8.5 mM
-chloroalanine, 0.2 mM EDTA, 0.4 mM MgCTP, 0.28 M sorbitol, 0.27 M
mannitol, 50 µM L-serine, 3 µCi of
[3H]serine, and the permeabilized cells equivalent to 90 µg of protein in 100 µl per reaction. The purpose of
-chloroalanine is to inhibit extraneous metabolism of serine to
sphingolipids. The assay mixture was incubated at 30 °C for 100 min
unless stated otherwise. The radiolabeling reaction was stopped by the
addition of 1 ml of chloroform, 1 ml of methanol, and 0.9 ml of 0.2 M KCl. The organic phase was further washed with 1.9 ml of
methanol/PBS (1:0.9, v/v) twice. The resulting chloroform phase was
dried under nitrogen gas, resuspended in 30 µl of chloroform/methanol
(9:1, v/v), and then loaded on silica gel H thin layer chromatography
plates. The plates were developed using a solvent system containing
chloroform, methanol, 2-propanol, 0.25% aqueous KCl, triethylamine
(30:9:25:6:18, v/v/v/v/v). Individual phospholipids were identified
under ultraviolet light with authentic standards, by spraying the thin
layer plates with 0.1% aqueous 8-anilino-1-naphthalenesulfonic acid.
The lipid spots then were scraped into vials containing scintillation
fluid, and the amount of 3H was quantified.
Preparation of Processed Donors--
Thawed permeabilized cells
were centrifuged at 1000 × g for 5 min at 4 °C. The
resulting low speed supernatant (LSS) was collected for reconstituting
PtdSer transport or for isolating high speed supernatants (HSS) and
high speed membranes (HSM) as described below. The remaining
permeabilized cell pellet was washed twice with 25-fold volume of cold
lysis buffer. After the second wash, the cells were resuspended in cold
lysis buffer to approximately the original volume present when they
were first thawed and used as the processed donors. In the indicated
experiments, washed donors were preincubated with 50 µM
CDP diacylglycerol (CDP-DAG), at 30 °C for 20 min. Excess CDP-DAG
was removed by 1000 × g centrifugation for 5 min at
4 °C, followed by washing the cells twice with a 20-fold volume of
cold lysis buffer. Prelabeled donors containing [3H]PtdSer were prepared from washed permeabilized cells
incubated in the PtdSer transport assay reagents, in the presence of
either MgCTP or CDP-DAG at 30 °C for 45 min. Unincorporated
radiolabel was removed by centrifugation and washing with cold lysis
buffer as described above for CDP-DAG-treated donors.
Preparation of LSS, HSS, and HSM--
Cellular components
released from permeabilized cells were fractionated by centrifugation.
LSS was collected as described above and further centrifuged at
400,000 × g for 1 h at 4 °C to obtain HSS and
HSM pellet. The membrane pellet was resuspended by homogenization in
cold lysis buffer using a battery-driven minihomogenizer (Kontes).
Analysis of Organelle Markers--
Fractions obtained from
permeabilized cells, including processed donors, LSS, HSS, and HSM,
were subjected to organelle marker analysis by either enzyme assays or
by antigen detection. The ER marker, PtdSer synthase (Pssp), was
measured by the method described by Carman and Bae-Lee (11). The
vacuolar marker, H+-ATPase, was detected by enzyme-linked
immunosorbant assays (12). The late Golgi marker, Kex2p protease, was
determined by using Boc-Gln-Arg-Arg-7 amidomethyl coumarin as the
substrate (13). Psd2p activity was determined as described previously
(14).
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RESULTS |
PtdSer Is Synthesized and Transported to the Locus of Psd2p in
Permeabilized Yeast--
In intact cells, PtdSer is synthesized in the
ER and related membranes and then transported to multiple organelles.
Upon arrival at the Golgi/vacuole, the PtdSer is decarboxylated by
Psd2p to form PtdEtn. PtdEtn can also be formed from nascent PtdSer by the action of Psd1p in the mitochondria. In a psd1
genetic background, all decarboxylation of PtdSer occurs via the action
of Psd2p (see Fig. 1). We permeabilized
psd1
cells and optimized conditions for the formation of
PtdEtn by Psd2p in the Golgi/vacuole. As shown in Fig.
2, the permeabilized strain, PTY40,
efficiently synthesizes [3H]PtdSer during the first 40 min of incubation, after which the level remains relatively constant,
probably reflecting the consumption of endogenous CDP-DAG required for
the reaction. Following a 20-30-min lag, the synthesis of
[3H]PtdEtn proceeds at a linear rate between 30 and 100 min. At 100 min, ~6% of the nascent [3H]PtdSer has
been converted to PtdEtn. A preliminary description of this system (7)
identified the Golgi and a novel light membrane fraction as the sites
of formation/accumulation of [3H]PtdEtn. These
experiments provide a clear demonstration that PtdSer transport to the
Psd2p can be reconstituted in permeabilized cells, making the process
amenable to further powerful analysis by biochemical and genetic
approaches.

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Fig. 1.
Biosynthesis and transport of
aminoglycerophospholipids in yeast. PtdSer is synthesized in the
endoplasmic reticulum and subsequently transported to other organelles.
In the mitochondria, the PtdSer is decarboxylated to form PtdEtn by
PtdSer decarboxylase 1. In the Golgi/vacuole compartments, PtdEtn is
formed by the action of PtdSer decarboxylase 2. PtdEtn in the
mitochondria and Golgi/vacuole compartments can be subsequently
exported from these organelles back to the endoplasmic reticulum for
further metabolism to PtdCho by the action of PtdEtn
methyltransferases. Both known and proposed mutations in the metabolic
and transport process are shown in lowercase
italic type: pss, PtdSer synthase;
pstA, PtdSer transport A pathway; pstB, PtdSer
transport B pathway; pstB1, the PtdIns-4 kinase, Stt4p;
pstB2, a PtdIns transfer protein; psd1, PtdSer
decarboxylase 1; psd2, PtdSer decarboxylase 2;
peeA, PtdEtn export A pathway; peeB, PtdEtn
export B pathway; pem1, PtdEtn methyltransferase 1;
pem2, PtdEtn methyltransferase 2. The focus of this study is
upon events between PtdSer synthesis and decarboxylation by Psd2p. For
this report, psd1 mutants were used in all experiments,
and the decarboxylation by the mitochondrial enzyme was completely
blocked as indicated by the X through the
mitochondria.
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Fig. 2.
Permeabilized yeast synthesize and transport
PtdSer to the site of Psd2p. PtdSer transport was monitored by
following the sequential incorporation of [3H]serine into
PtdSer and PtdEtn in permeabilized cells containing a
psd1 allele as described under "Experimental
Procedures." Duplicate 100-µl aliquots were taken at each indicated
time point, and radiolabeled phospholipids were extracted and analyzed
by thin layer chromatography and liquid scintillation spectrometry.
Data shown are means ± S.E. of at least four independent
experiments.
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PtdEtn Formation Requires Cellular Components Released from
Permeabilized psd1
Yeast--
Following permeabilization, some
cellular components, including cytosol and membranes, were released
from the cells. The released components were initially separated from
the permeabilized cells by a low speed centrifugation. We next examined
the role of the released material in PtdSer transport and
decarboxylation. Permeabilized cells, washed free of released cellular
contents by a 1000 × g centrifugation, lost their
ability to transport PtdSer to Psd2p (Fig.
3A). However, the transport
activity was restored when the cells were reconstituted with their
autologous LSS (Fig. 3B). The relative efficiency of PtdEtn
formation after reconstitution with LSS approximated that of the
unmanipulated cells. Both the rate and extent of PtdEtn formation by
LSS-supplemented permeabilized cells were comparable with the results
obtained in the experiments shown in Fig. 2.

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Fig. 3.
PtdSer transport can be reconstituted with
washed permeabilized cells and LSS. Washed permeabilized cells
were combined with a volume of LSS equivalent to that removed by
centrifugation and added to the PtdSer synthesis/transport reaction
mixture. PtdSer transport was followed by taking a 100-µl aliquot
from the reaction mixture at the indicated time points, extracting the
lipids, and analyzing them by thin layer chromatography and liquid
scintillation spectrometry. PtdSer transport activity is expressed as
the percentage of radiolabel incorporated into PtdEtn relative to the
total radiolabel present in PtdSer plus PtdEtn at the end of a 100-min
incubation. A, washed permeabilized cells alone;
B, washed permeabilized cells reconstituted with LSS. The
results are an average of two independent experiments performed in
duplicate.
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The synthesis of PtdSer in the permeabilized cells requires a CDP-DAG
precursor (8). Supplementation of the permeabilized cells with CTP in
the standard reaction described in Fig. 2 relies upon the action of
CDP-DAG synthase as well as endogenous CDP-DAG to drive the PtdSer
synthase reaction. With prolonged incubation, it is likely that the
generation of CDP-DAG by the corresponding synthase will deplete the
requisite phosphatidic acid pool. To circumvent the reliance upon
phosphatidic acid and endogenous CDP-DAG pools, we examined the
efficacy of preincubating the washed permeabilized cells with CDP-DAG.
The results of these experiments are shown in Fig.
4. In these studies, we compared the
formation of PtdEtn in permeabilized cells, prepared as described in
Fig. 2, to that found from washed donors supplemented with MgCTP or CDP-DAG, in either the presence or absence of LSS. Under all conditions of reconstitution, the formation of PtdEtn is strongly dependent upon
the addition of LSS. Preincubation of washed donors with CDP-DAG
consistently gives better synthesis of PtdEtn. We believe this is due,
in part, to the more proximal nature of the reactions to PtdSer
formation compared with CTP supplementation. Most significantly, we
also find that preincubation of the washed donors with
[3H]serine and CDP-DAG followed by centrifugation to
remove precursors allows us to effectively pulse-label the PtdSer pool
in this compartment, thereby permitting the independent examination of
events occurring between PtdSer synthesis and PtdEtn formation.

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Fig. 4.
Washed donors prelabeled with
[3H]serine in the presence of CDP-DAG remain competent to
transport PtdSer in the presence of LSS. PtdSer transport by
permeabilized cells in the standard assay (Control) was
compared with that obtained by reconstitution with washed donors in
either the absence ( LSS) or presence (+LSS) of
LSS under the same reaction conditions. Additional comparisons were
made in which washed donors were preincubated with CDP-DAG for 45 min
and then washed to remove excess CDP-DAG before the addition of LSS
plus [3H]serine. In a different variation, donors were
preincubated with CDP-DAG plus [3H]serine for 40 min and
then washed by centrifugation to remove these precursors prior to
incubation with LSS. The reactions were terminated by lipid extraction,
and the lipids were analyzed by thin layer chromatography and liquid
scintillation spectrometry. The data are expressed as the percentage of
radiolabel in PtdEtn relative to that found in PtdSer plus PtdEtn at
the end of the incubation. The data are means ± S.E. for at least
three experiments.
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LSS Contains both Acceptor Membranes and Cytosol--
We next
sought to characterize the components present in LSS and the manner in
which they contribute to the transport dependent conversion of nascent
PtdSer to PtdEtn. Initially, we examined the LSS for the presence of
Psd2p and marker enzymes for the ER (Pssp), Golgi (Kex2p), and vacuoles
(H+ATPase). As shown in Fig.
5, Pssp was retained by the washed
permeabilized cells and absent from the LSS. The essentially complete
absence of Pssp from the LSS makes it an ideal acceptor compartment
because it is devoid of precursor synthesis. The LSS contains ~40%
of the recoverable Golgi, 30% of the recoverable vacuolar marker, and
51% of the recoverable Psd2p. The total recovery of all the organelle
markers between the processed permeabilized cell and its autologous LSS
is between 44 and 70%, implying that significant amounts of the
relevant enzymes in permeabilized cells may be either inactivated or
lost during the preparation of the washed donor compartment and the
LSS. Although significant amounts of the Golgi, the vacuole, and Psd2p
remained with the processed donors, nascent PtdSer was not efficiently
transported to these loci (Fig. 4). One likely explanation of this
finding is that the factors involved in the release of the acceptor
membranes may also be mechanistically relevant to the lipid
transfer process.

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Fig. 5.
Distribution and recovery of organelle marker
enzymes among fractions derived from permeabilized cells. The
distribution of organelle marker enzymes among permeabilized cells,
washed donors, LSS, HSS, and HSM was determined by assaying the
enzymatic activities or immunoreactivity of the indicated proteins:
Pssp from endoplasmic reticulum; Kex2p from Golgi apparatus; vacuolar
H+ATPase (Vac ATPase) from vacuole; Psd2p from
Golgi and vacuole. Recoveries in each fraction are expressed as the
percentage of values initially found in unwashed permeabilized cells
(100%). The results are means ± S.E. of eight individual
samples.
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Acceptor Membranes Alone Are Sufficient to Reconstitute PtdSer
Transport--
For further characterization, the LSS was subjected to
ultracentrifugation at 400,000 × g for 1 h to
generate HSM and HSS. The distribution of the ER, Golgi, and vacuolar
enzymes and Psd2p in the HSM relative to other fractions is shown in
Fig. 5. Almost all of the Golgi-Kex2p, vacuolar H+ATPase,
and Psd2p present in the LSS was recovered in the HSM. Essentially none
of these enzymes were recovered in HSS. This further partitioning of
LSS allowed us to test the roles of the acceptor membranes and the
cytosol individually. The specific questions that we wanted to address
were whether 1) cytosol contains factors that could stimulate PtdSer
transport between the donor membranes and the acceptor membranes that
remained in the processed permeabilized cells, 2) isolated acceptor
alone is sufficient to reconstitute PtdSer transport, and 3) both
cytosol and acceptor membranes are required for PtdSer transport. When
HSM alone, as the acceptor membrane, was reconstituted with processed
donors, it resulted in a concentration-dependent increase
of PtdSer transport activity as shown in Fig.
6. Due to the volume limitation of our assay, we were only able to test two concentrations of HSS. HSS alone
weakly stimulated PtdSer transport and was significantly less effective
than HSM alone. When both HSS and HSM were present to reconstitute
PtdSer transport, HSS modestly enhanced PtdSer transport at low HSM
concentration. However, the HSS effect was diminished when higher
concentrations of HSM were present to support PtdSer transport (Fig.
6). This result demonstrates that, although there may be some cytosolic
factors that can weakly stimulate PtdSer transport from the ER to the
locus of Psd2p, the addition of acceptor membranes (i.e.
HSM) alone, is sufficient to reconstitute the transport process in the
permeabilized cell assay system.

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Fig. 6.
HSM alone are sufficient to reconstitute
PtdSer transport and decarboxylation. Prelabeled, washed donors
derived from permeabilized cells equivalent to 90 µg of total protein
were examined for PtdSer transport in the absence or the presence of
the indicated amount of HSS or HSM or both. The "volume equivalent"
of HSS or HSM is equal to the indicated multiple of the amount of the
fraction initially isolated from 90 µg of permeabilized cell
proteins. PtdSer transport activity is reported as -fold increase of
PtdSer transport relative to that found for prelabeled donors alone.
Results are means ± S.E. of three individual experiments.
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Transport of Nascent PtdSer to Psd2p Is
Nucleotide-independent--
Achleitner et al. (8) showed
that PtdSer transport to yeast mitochondria is independent of ATP.
However, PtdSer translocation in permeabilized mammalian cells has been
demonstrated to be ATP-dependent (15-17). In addition, ATP
as well as GTP are also involved in the trafficking and docking of
vesicles effecting interorganelle protein transport (18-20). We
examined whether the translocation of PtdSer from the ER to the Psd2p
locus in yeast is nucleotide-dependent. The addition of
Mg2+ salts of ATP, ADP, CTP, or UTP (at 1 mM)
or of GTP, GDP, or GTP
S (at 0.2 mM) failed to
significantly alter PtdSer transport reconstituted between the donor
membranes and acceptors. These findings demonstrate that our
reconstituted system does not require nucleotides as cofactors for
PtdSer transport.
EDTA Inhibits PtdSer Transport to the Psd2p-containing Acceptor
Membranes--
Previously, only a trace amount of PtdSer transport to
the Psd2p locus could be detected in the permeabilized
psd1
cells (8). As described under "Experimental
Procedures," one alteration we made to optimize the measurement of
PtdSer translocation to Psd2p was to omit EDTA from the incubations.
Our success raised the question of whether PtdSer movement to the
Golgi/vacuole Psd2p is sensitive to EDTA. Indeed, the addition of EDTA
not only inhibited nascent PtdSer synthesis but also arrested further
conversion of PtdSer into PtdEtn in unmanipulated permeabilized cells
(Fig. 7). This inhibitory effect is not
due to the inhibition of catalysis of Psd2p, since the enzyme does not
require divalent cations for activity and is unaffected by the presence
of EDTA (4). One possible explanation is that ongoing PtdSer synthesis
is required for continued PtdSer transport, and the chelation of the
Pssp cofactor, Mn2+ ions, by EDTA thus arrests both
activities. However, this explanation is unlikely, since reconstituted
PtdSer transport, using processed donors with a prelabeled PtdSer pool
(Figs. 4 and 6), bypasses continuous PtdSer synthesis. The elimination
of the two possibilities described above implies that EDTA may inhibit
the PtdSer transport process directly. To test this hypothesis, we
measured reconstituted PtdSer transport activity with prelabeled donors
either in the absence or the presence of Mn2+ ions. The use
of prelabeled donors allows us to exclude any effects on Pssp activity
and restricts the conclusions to events occurring after PtdSer
formation. As shown in Fig. 8, the
transport activity was dependent upon Mn2+ ions. In
addition, the transport activity was abolished when EDTA was added
together with Mn2+. These results demonstrate a specific
requirement for a divalent cation in the reconstituted transport
reaction.

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Fig. 7.
EDTA inhibits transport of PtdSer to Psd2p in
unmanipulated permeabilized cells. PtdSer transport was measured
using unwashed permeabilized cells as described under "Experimental
Procedures" and in the legend to Fig. 2. After 45 min of incubation,
either 4 mM EDTA (arrow) or buffer was added to
samples, and the reactions were continued for an additional 55 min.
Samples of 100 µl were removed from the reactions at the indicated
times, and the extracted lipids were analyzed by thin layer
chromatography and liquid scintillation spectrometry. A and
B show the incorporation of radiolabel into PtdSer and
PtdEtn, respectively. Data shown are from two experiments performed in
duplicate.
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Fig. 8.
EDTA inhibits transport of PtdSer from
prelabeled donors to HSM acceptors. PtdSer transport was
reconstituted utilizing prelabeled donors and 2× volume equivalents of
HSM in the presence or the absence of 0.6 mM
MnCl2 and/or 4 mM EDTA. PtdSer transport
activity is reported as the -fold increase in PtdSer transport relative
to prelabeled donors. Results shown are means ± S.E. of 3-6
individual experiments.
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PtdSer Transport Requires Manganese Ions--
Although
Mn2+ was the only divalent cation added to our
reconstituted washed donor/acceptor assay system described in Fig. 8, it does not elucidate whether this is a general or specific requirement for cations as cofactors for PtdSer transport. Therefore, we tested a
broad range of divalent cations for their ability to stimulate PtdSer
transport. The additional divalent cations examined included Mg2+, Cu2+, Ni2+, Zn2+,
and Ca2+. With the exception of Ca2+, all
divalent cations were tested at 1 mM. Because 1 mM Ca2+ caused extensive degradation of the
labeled PtdSer pool, 50 µM Ca2+ was tested.
The degradation of PtdSer by the addition of calcium may be due to the
activation of a calcium-dependent phospholipase (21, 22).
Among all of the ions examined, only Mn2+ ions supported
PtdSer transport activity. These findings demonstrate that
Mn2+ plays a unique role as a cofactor in reconstituted
PtdSer transport.
The Acceptor Membrane Pool Requires Elements Other than
Psd2p--
Because our assay utilizes the product of the Psd2p
reaction as our transport signal, one could argue that the apparent
dose-dependent transport activity of HSM is simply due to
the increasing amount of Psd2p activity added to the assay system and
does not truly reflect a transport process. To investigate whether such
an argument is valid, we compared HSM isolated from permeabilized
psd1
and psd1
/pstB2
cells. We
have previously reported that the PSTB2 gene encodes a
protein involved in the transport-dependent metabolism of
PtdSer (7). Strains harboring psd1
/pstB2
mutations have normal Psd2p activity but are defective in converting
PtdSer into PtdEtn in both intact and permeabilized cells (7). As shown in Fig. 9, HSM from permeabilized
psd1
/pstB2
cells containing amounts of
Psd2p activity comparable with HSM from psd1
strains failed to reconstitute PtdSer transport. Most importantly, the addition
of excess Psd2p from psd1
/pstB2
strains is
completely ineffective at overcoming any of the transport defect. This
result indicates that the transport activity of HSM does not solely
depend on the Psd2p activity but clearly involves other factors that have been specifically implicated in lipid transport by in
vivo biochemical and genetic experiments. This latter finding
emphatically demonstrates that the permeabilized cell system
recapitulates processes found with intact cells.

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Fig. 9.
The reconstituted transport system requires
factors other than Psd2p to decarboxylate donor-derived PtdSer.
Prelabeled psd1 donors were reconstituted with various
amounts of HSM isolated from either psd1 or
psd1 /pstB2 permeabilized cells. The Psd2p
activity of either HSM was independently determined by in
vitro assays utilizing
1-acyl,2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl)aminocaproyl-Ptd-[1'-14C]serine
as the substrate. One unit of Psd2p activity is defined as the
production of 1 pmol of 14CO2/min. PtdSer
transport activity is reported as the -fold increase of PtdSer
transport signal relative to the prelabeled donors alone. The results
are means ± S.E. of three independent experiments.
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The Permeabilized Cell System Localizes the pstB2 Defect to the
Acceptor Membrane Compartment--
The experiments described above do
not permit localization of the pstB2 defect. However, the
ability to segregate the components of the permeabilized cell system
affords the opportunity to critically test whether the pstB2
defect resides in the donor or the acceptor compartment. In these
experiments, donors and acceptors were prepared from cells with either
wild type or null PSTB2 alleles. The wild type donors were
tested for their ability to transport nascent [3H]PtdSer
to either wild type or mutant acceptors. Conversely, pstB2
donors were also tested for their ability to
transfer [3H]PtdSer to either wild type or mutant
acceptors. As shown in Fig. 10, the
wild type donors could transfer PtdSer to wild type acceptors but not
pstB2
acceptors. When pstB2
donors were
used as a source of [3H]PtdSer, the lipid was readily
transferred to wild type acceptors but not mutant acceptors. These
results clearly demonstrate that the pstB2
-harboring
strains produce competent donor membranes but incompetent acceptor
membranes.

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Fig. 10.
The pstB2 mutation affects
acceptor membrane competence. Washed, prelabeled donor and
acceptor (HSM) membranes were prepared as described in the legend to
Fig. 4 using psd1 strains with either wild type
(PSTB2) or mutant alleles (pstB2 ) at the
second genetic locus. The donors were incubated without HSM or with HSM
derived from either the PSTB2 or pstB2 strain.
PtdSer transport activity was measured as the -fold increase in PtdEtn
formation relative to that obtained from prelabeled donors alone.
Results are means ± S.E. from three experiments.
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DISCUSSION |
The heterogeneity of the lipid composition of different organelle
membranes has been known as a general principle of cell biology for
decades (23). However, the molecular mechanisms and gene products
responsible for generating this membrane diversity remain poorly
defined. We have focused on the lipid aspect of membrane biogenesis by
examining aminoglycerophospholipid synthesis in Saccharomyces
cerevisiae. We have conducted both biochemical and genetic
approaches to this problem in an effort to identify new genes and their
products and mechanisms of actions. In this report, we describe the
successful reconstitution of PtdSer transport between the ER or closely
related membranes and Psd2p.
The permeabilized cell system that we developed shows
time-dependent synthesis of PtdSer and PtdEtn. The level of
nascent [3H]PtdSer formed plateaus after 40 min of
incubation presumably due to the depletion of phosphatidic acid and
CDP-DAG pools. The formation of [3H]PtdEtn proceeds after
a lag of 20-30 min. This lag period is likely to comprise some feature
of PtdSer movement between elements of the ER and the Golgi
apparatus. Recent studies have demonstrated that most of the nascent
[3H]PtdEtn is found in the Golgi and a novel light
membrane fraction (7). Although 70% of the Psd2p activity colocalizes
with the vacuole compartment (7), we do not see the appearance of
[3H]PtdEtn in this organelle in the permeabilized cell
system (7). These findings may mean that the permeabilization procedure
destroys the machinery for PtdSer transport to the vacuole.
Alternatively, the results may indicate that there is interorganelle
cooperation between the Golgi and the vacuole with respect to the pool
of Psd2p available for use in transport-dependent lipid
metabolism. The formation of [3H]PtdEtn provides a
relatively strong biochemical signal for PtdSer transport. This signal
was probably missed in earlier studies with permeabilized cells as a
consequence of inadequate Mn2+ in the reaction. We estimate
that the rate of PtdEtn formation is ~20% of that occurring in
intact cells.
Low speed centrifugation and washing of permeabilized cells resolves a
sedimentable donor compartment capable of PtdSer synthesis but only
weakly competent for decarboxylation. The LSS fraction restores the
transport dependent metabolism of PtdSer to PtdEtn to the washed
donors. In general, reconstitution of washed donors with LSS restores
PtdEtn formation to levels between 50 and 100% of those found for
unprocessed permeabilized cells. The ability to physically isolate the
donor compartment permitted optimization of the conditions for
synthesis of the PtdSer precursor. Preincubation of the donors with
[3H]serine and CDP-DAG provided a convenient and
efficient method for radiolabeling the PtdSer pool in the donors. Most
importantly, this approach allowed for both temporal and physical
segregation of PtdSer synthesis from its transport dependent
decarboxylation. As a consequence of this refinement of the
permeabilized cell system, we are more accurately able to assess
requirements for events occurring after PtdSer synthesis.
Subfractionation of the LSS into HSS and HSM components provides
additional important details about the minimal requirements for
reconstitution of PtdSer transport. These experiments provide clear
evidence for the existence of two pools of Psd2p. One pool of Psd2p is
retained within the permeabilized cell and functions relatively
inefficiently in the decarboxylation of nascent PtdSer. In contrast, a
pool of Psd2p released from the cells is more effective at the
decarboxylation of PtdSer. We currently understand few of the details
that characterize these two compartments containing Psd2p. However, we
propose that elements of the system that are permissive for the release
of Psd2p may be important components affecting PtdSer transfer between organelle domains. The current studies clearly demonstrate that the HSM
derived from LSS is fully competent to import PtdSer and decarboxylate
it. Varying HSM shows that the rate of PtdEtn formation is dependent
upon the amount of acceptor added.
The reconstituted transport system composed of either permeabilized
cells or washed donor/acceptor membranes displays marked sensitivity to
chelation of Mn2+ with EDTA. Upon the addition of EDTA, the
formation of PtdEtn is immediately arrested. The requirement for
Mn2+ appears specific, since other common divalent cations
fail to substitute for its function. The Mn2+ requirement
appears to be an uncommon aspect of lipid transport that may
distinguish the process from interorganelle protein transport.
Comparison of reconstituted transport using HSM derived from
psd1
strains and psd1
/pstB2
strains provides compelling evidence that the PtdSer transfer to the
locus of Psd2p is specific. In vivo studies have provided
clear evidence that pstB2 mutants are profoundly defective
in decarboxylation of nascent PtdSer despite the presence of wild type
levels of Psd2p. This same defect is reproduced with permeabilized
psd1
/pstB2 cells. The reconstituted donor/acceptor system reveals that even in the presence of excess Psd2p
in HSM, there is no significant decarboxylation of nascent PtdSer by
cells harboring a pstB2 mutation. This latter result effectively rules out the occurrence of nonspecific fusion and artifactual decarboxylation in the permeabilized cell system.
The reconstituted transport system also allows for assignment of the
subcellular site of action of PstB2p. Previous studies have
demonstrated that PstB2p is an amphitropic protein present in both the
cytosol and multiple membranes (7). The experiments described in this
report demonstrate that donor membranes prepared from
pstB2
mutant cells are fully competent to transfer
nascent PtdSer to wild type acceptor membranes for the formation of
PtdEtn. In contrast, wild type donor membranes cannot effectively
transfer nascent PtdSer to Psd2p present in the pstB2
acceptor membranes. Thus, the pstB2
defect clearly
resides in the acceptor membranes. These findings are consistent with
PstB2p regulating the final steps of substrate access to the PtdSer decarboxylase.
In summary, we have defined a powerful new biochemical system for
examining PtdSer transport events between the ER and the Golgi
apparatus. This system should prove amenable to further dissection and
reconstitution. We anticipate that this system will also facilitate the
identification of the sites of function of new gene products in the
lipid transport process and provide a biochemical assay system for
elucidating their mechanism of action.