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INTRODUCTION |
The biogenesis of organelle membranes requires the coordinated
transport of proteins and phospholipids from their sites of synthesis
to their final locations. Considerable molecular information is
available regarding protein sorting for assembly into membranes (1, 2).
In contrast, little is known about the mechanisms required for lipid
transport within eukaryotic cells. Multiple mechanisms for lipid
transport have been proposed including (a) vesicle packaging
and routing, (b) phospholipid transfer proteins, and
(c) zones of apposition between donor and acceptor organelle compartments (3, 4). Some experimental evidence exists in support of
each of the above proposed mechanisms. However, the current results
suggest considerable diversity, rather than a simple set of unifying
principles, is involved in the process of lipid transport.
In an effort to address the problem of lipid transport, this laboratory
has focused upon the metabolism of
PtdSer1 in the yeast
Saccharomyces cerevisiae (5-9). The lipid, PtdSer, provides
a number of advantages for examining transport because the multiple
events in its conversion to phosphatidylcholine (PtdCho) occur within
different organelle domains and provide discrete biochemical indicators
of transport steps. The topology of PtdSer metabolism and the basis of
the genetic strategy used in this study are outlined in Fig. 1. PtdSer
is synthesized in the endoplasmic reticulum or closely related
membranes by PtdSer synthase (10). Following its synthesis, the lipid
is disseminated throughout the cell. Upon arrival at the mitochondria
or the Golgi/vacuole, PtdSer is decarboxylated to form PtdEtn (8, 11).
The mitochondrial decarboxylase is Psd1p, and the Golgi/vacuole enzyme
is Psd2p (5, 7). Following its formation in either the mitochondria or
the Golgi/vacuole, PtdEtn is transported to the endoplasmic reticulum
for the synthesis of PtdCho (5, 12). The methyltransferase enzymes
Pem1p and Pem2p catalyze the formation of PtdCho from PtdEtn using
S-adenosylmethionine as the methyl donor (13).
In this study, we have used cells harboring the null allele,
psd1
::TRP1, to force all PtdSer metabolism
through the Golgi/vacuolar compartments (5, 9). In this genetic
background, we reason that it should be possible, after mutagenesis, to
isolate new strains with defects in metabolic steps between PtdSer
formation and PtdEtn methylation. Included in these steps are vectorial lipid transport events between the endoplasmic reticulum and the Golgi/vacuole for PtdSer and between the Golgi/vacuole and endoplasmic reticulum for PtdEtn. Our approach follows from the observations that
cells with inactivating mutations in PtdSer synthase (PSS) or both PSD1 and PSD2 genes remain viable if
supplemented with ethanolamine (Etn) (7, 8, 12). Etn rescues the
aforementioned strains by providing PtdEtn via the CDP-ethanolamine
pathway. These latter findings imply that specific defects in PtdSer
and PtdEtn transport may be identifiable and amenable to rescue by Etn
supplementation. This general approach has been applied in a previous
study and has identified the PtdIns 4-kinase, Stt4p, as an important
component in PtdSer metabolism in steps between its synthesis in the
endoplasmic reticulum and decarboxylation in the Golgi/vacuole (9). Our
goals in this study were to: 1) identify new Etn auxotrophs among
mutagenized populations of strains with a
psd1::TRP1 allele, 2) characterize the Etn
auxotrophs genetically and biochemically, and 3) identify and
characterize the gene complementing the defect.
In this report we identify a new strain, designated pstB2,
with interesting properties in aminoglycerophospholipid metabolism. The
strain is an Etn auxotroph with pronounced defects in phospholipid metabolism. The defects in lipid metabolism include the accumulation of
high levels of PtdSer in the Golgi fraction and a light membrane fraction. The gene complementing the growth and biochemical defect encodes a protein of previously unknown biochemical function with structural similarity to the PtdIns/PtdCho transfer protein, Sec14p.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All chemicals, including amino acids for yeast
media, were purchased from either Sigma or Fisher Scientific. Other
components for yeast and bacterial growth media were purchased from
Difco. Phospholipids were obtained from Avanti Polar Lipids. Thin layer silica gel H plates were purchased from Analtech Corp. The
radioisotopes [3-3H]serine and
[1-14C]serine were from Amersham Pharmacia Biotech and
ICN, respectively. The lipid
1-acyl,2-(6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl-Ptd-[1'-14C]Ser
(referred to as NBD-Ptd-[1'-14C]Ser) was synthesized from
[1-14C]serine and NBD-CDP-diacylglycerol using
Escherichia coli PtdSer synthase (14). Reagents for protein
determination were either from Pierce or Bio-Rad. Pre-cast
SDS-polyacrylamide gels were purchased either from NOVEX or FMC. Mouse
monoclonal antibodies against the V5 epitope tag of the PstB2p fusion
protein were obtained from Invitrogen. Other reagents used for ligand
blotting were obtained from Bio-Rad and Sigma.
Cells, Plasmids, and Libraries--
Yeast were cultured in
synthetic complete or YPDAUE (standard YPD medium plus additional
adenine, uracil, and ethanolamine) media (9). When the PstB2p fusion
protein was overexpressed in yeast under GAL1 promoter
regulation, the cells were grown in uracil-free synthetic medium
containing ethanolamine, with 2% galactose and 1% raffinose as the
carbon sources. The parental strain, RYY52 (MAT
lys2 trp1 ura3 his3 leu2 suc2 psd1-1::TRP1), was
constructed from the wild type strain, SEY6210 (MAT
lys2 trp1 ura3 his3 leu2 suc2), as described previously (5). The psd2 strain, PTY43 (MATa ura3 leu2 his3 trp1 ade2
suc2 psd2-1::HIS3), used to examine complementation
by the ethanolamine auxotrophs was constructed directly from wild type
SEY6211 (MATa ura3 leu2 his3 trp1 ade2 suc2) as
described previously (8). The pss mutant strain PTY70
(MATa his3 cho1-1) used to identify mutants carrying a defect in
PSS by complementation analysis was obtained from a cross of
KA101, generously provided by Dr. Susan A. Henry (Carnegie Mellon University, Pittsburgh, PA) (12), with X1049 (Yeast
Genetic Stock Center). In this paper we use the simplifying terminology PSS and pss, respectively, to identify the wild
type and mutant alleles of phosphatidylserine synthase in place of the
original CHO1 and cho1 designations (15-17). The
pstB2 mutant strain, WWY54, was isolated as an
ethanolamine auxotroph from the ethylmethane sulfonate-mutagenized (18)
parental strain RYY52. RYY57 and WWY71 are two
psd1::TRP1 strains used to outcross the
original pstB2 mutant. WWY71 was obtained from a
cross between PTY40 (MATa ura3 his3 trp1 met14
psd1-1::TRP1) and PTY41 (MAT ura3
leu2 trp1 his3 lys2 psd1-1::TRP1). The
sec14 strain used was CTY1-1A (MATa ura3-52
his3-200 lys2-801 s14-3ts).
The YCp50 yeast genomic library was generously provided by Dr. Vytas
Bankaitis (University of Alabama, Birmingham, AL). The YEp352 plasmid
was obtained from Dr. Alex Franzushoff (University of Colorado Health
Sciences Center, Denver, CO). The pUC18-HIS3 construct was a
gift from Dr. Rodney Rothstein (Columbia University, NY).
Preparation of the Yeast Total Membranes and
Microsomes--
Yeast cell-free extract was prepared as described
previously (9). The total membranes were collected from the cell-free extract by ultracentrifugation at 100,000 × g for
1 h at 4 °C. The resulting membranes were resuspended using a
Potter-Elvehjem device. The microsomes were also prepared from the
cell-free extract by centrifugation at 15,000 × g for
20 min to remove dense membranes followed by ultracentrifugation at
100,000 × g for 1 h at 4 °C to sediment the
low density membranes. The protein concentrations of the total
membranes and the microsomes were determined using a bicinchoninic acid
method (Pierce) or the Bradford method (19).
Enzyme Assays--
PSD2 activity was measured as described
previously (8). Briefly, the activity was assayed by trapping
14CO2 released from NBD-Ptd
(1'-14C]Ser on 2 M KOH-impregnated filter
paper at 30 °C. PtdSer synthase activity was determined by
monitoring the incorporation of either [3H]serine or
[14C]serine into PtdSer as described by Carman and
Bae-Lee (20).
Radiolabeling and Phospholipid Analysis of Cells--
All
strains were grown in synthetic complete medium plus ethanolamine at
30 °C until reaching mid-log phase. Cultures were then washed and
diluted to an A600 nm of 0.2 in a volume of 2 ml
with synthetic complete medium (SC) containing 10 µCi/ml
L-[3H]serine (50 µCi/nmol). These cultures
were incubated at 30 °C with shaking for 4 h. Trichloroacetic
acid (5% (w/v) final concentration) was added to each culture at the
end of the incubation, and cells were washed twice with ice-cold water.
Lipids were isolated by ethanol extraction (21) and analyzed by thin
layer chromatography (TLC) on Silica Gel H plates using a solvent
system containing chloroform, methanol, 2-propanol, 0.25% KCl,
triethylamine (30:9:25:6:18, v/v). Phospholipids were identified by
co-chromatography with authentic standards and visualized by staining
the TLC plates with 0.1% 8-anilino-1-naphthalene sulfonic acid and
exposure to ultraviolet light. Individual lipid spots were scraped from
the TLC plates into 0.5 ml of water plus 4.5 ml of scintillation
mixture (Fisher Scientific). The radioactivity of each lipid sampled
was determined in a liquid scintillation counter.
Isolation of the PSTB2 Gene--
The Etn auxotrophic mutant,
pstB2, was transformed with a YCp50 yeast genomic library
using the YEASTMAKER kit purchased from CLONTECH.
Cells harboring plasmids were first selected for the presence of the
plasmid marker, URA3, by growth on synthetic complete medium
plus ethanolamine lacking uracil. The transformants were then screened
for ethanolamine prototrophic growth on SC medium lacking uracil. The
complementing plasmids were recovered from the positive transformants
and amplified in E. coli as described previously (22).
Amplified plasmids were isolated from the E. coli and
retransformed into the pstB2 mutant to confirm their ability
to complement the growth defect of the mutant strains. Further
confirmation of the activity of the complementing plasmid was obtained
from experiments that demonstrated concordant loss of Etn prototrophy
and the linked plasmid uracil prototrophy. For plasmid loss studies,
the pstB2 mutant carrying complementing plasmids was
cultured under nonselective conditions for greater than 30 generations
and then monitored for both ethanolamine and uracil auxotrophy.
DNA Sequencing and Plasmid Constructs--
The original YCp50
PSTB2 clones were sequenced from both ends of the insert using primers
that annealed to the plasmid sequences adjacent to opposite ends of the
insertion site. Sequencing was performed utilizing the ABI prism Ready
Dye Deoxy Terminator Cycle Sequencing Kit. Cycle sequencing products
were purified with Centricep Spin Columns (Princeton Separations,
Adelphia, NJ) and then analyzed on an ABI 377 automated sequencer at
the Molecular Resource Center of the National Jewish Medical and
Research Center. The resulting sequences were used to identify the
genomic location of the insert by searching the S. cerevisiae Genome Data base (23). Predicted open reading frames
(ORFs) within the insert were either individually subcloned or
subcloned as a cluster containing two to three ORFs into a YCp50 or
YEp352 vector depending on the available restriction sites. The
subclones were retransformed into the pstB2 mutant to test
their complementation of the Etn auxotrophy.
The pYES plasmid encoding a galactose inducible, V5His6
epitope-tagged form of PstB2p (PstB2V5His6p) was obtained
from Invitrogen and used for expression in yeast. A plasmid encoding
the epitope-tagged version of the protein for expression in insect
cells was constructed by excising the DNA encoding
PstB2V5His6p from the pYES plasmid using SspI
and XbaI restriction enzymes and ligating the fragment into
PCR 2.1. Ligation of the DNA into PCR 2.1 was performed after A-tailing
the insert with Taq polymerase. The desired insert was excised by EcoRI digestion and subsequently ligated into a
pVL1392 vector for expression in Sf9 cells.
Disruption of Chromosomal PSTB2 Allele--
The PSTB2 gene was
disrupted in a YEp352 vector by inserting a 1.8-kb BamHI DNA
fragment containing the HIS3 gene at the BclI site within the PSTB2 coding region. The
pstB2::HIS3 allele was removed from YEp352 by
SacI/XbaI digestion and then subcloned into the
E. coli vector pGEM4Z. The chromosomal PSTB2 gene
was disrupted by one-step gene replacement (24) using the YEASTMAKER system (CLONTECH) with the linear
SacI/XbaI fragment containing pstB2::HIS3 from the pGEM4Z plasmid construct.
Recombinants were selected for the presence of His+ prototrophy.
The disruption of the chromosomal PSTB2 gene was confirmed
by PCR (25). Primers flanking the disruption or complementing the
HIS3 marker gene were constructed such that the primer pairs annealed to the 5'- and 3'-end of the PSTB2 gene would yield
a 1.9-kb fragment for the wild type allele, whereas the HIS3
internal primer and the 3'-flanking primer of PSTB2 gene
would result in a 1.5-kb fragment for the disrupted allele. The
transforming DNA did not contain the sequence corresponding to the 5'-
or the 3'-PCR primer to ensure that the appropriate PCR products could
only be generated after integration into the PSTB2 locus.
Genomic DNA was prepared from strains by standard methods (26), and the PCR reaction was performed using the Taq DNA polymerase in a
Perkin-Elmer DNA Thermalcycler. Amplified fragments were visualized by
agarose gel electrophoresis and staining with ethidium bromide
(27).
PtdIns and PtdSer Transfer Assays--
Both Sec14p and PstB2p
were overexpressed as amino-terminal His6 fusion proteins
under control of the lac operator in E. coli using a pQE30
vector (Qiagen). An E. coli lysate containing the fusion
protein was prepared from
isopropyl-1-thio-
-D-galactopyranoside-induced cells by
sonicating in lysis buffer (50 mM sodium phosphate, pH 7.4, 0.3 M NaCl. 2 mM -mercaptoethanol, 1 mM NaN3, 7 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, and 7 µg/ml DNase
I) on ice for a total of 3 min in 1 min bursts with 1 min pauses on ice
between each burst. The resulting homogenate was centrifuged at
3000 × g for 10 min followed by a 30,000 × g centrifugation for 20 min at 4 °C to obtain cytosol
containing His6PstB2p or His6Sec14p. PtdIns and
PtdSer transfer activity of the recombinant His6PstB2p was
determined as described previously (28, 29).
Expression of the Epitope-tagged PSTB2 Fusion Protein in Yeast
and Sf9 Cells--
The PSTB2 gene was expressed in
yeast under the GAL1 promoter as a fusion protein with a
V5His6 epitope at its 3'-end. Epitope-tagged PstB2p was
expressed in the pstB2 strain grown in SC medium containing galactose and raffinose as the carbon sources. Cells were harvested at
mid-log phase. For expression in Sf9 cells a pVL1392 construct harboring the coding sequence of PstB2V5His6p was
cotransfected with BaculoGoldTM Autographa
californica DNA (Pharmingen) into a monolayer of Sf9 cells
using the CaCl2 method. The Sf9 cells were routinely
grown in Trichoplusia ni medium (30) containing 10%
heat-inactivated fetal bovine serum. General procedures for the growth,
maintenance, and infection of Sf9 cells followed the methods
described by O'Reilly et al. (30). For expressing
PstB2V5His6p, 4 × 107 Sf9 cells
grown in 150-mm dishes were infected at a viral multiplicity of 10 for
60 h. The cells were gently scraped off the culture dish and
collected by centrifugation at 4 °C. The harvested cells were washed
twice in cold phosphate-buffered saline (PBS). The final cell pellet
was snap frozen over dry ice and stored at 
80 °C. The uninfected
Sf9 cells or cells infected with recombinant virus containing
the S. cerevisiae DPP1 gene (31) were used as controls.
Detection of the Epitope-tagged PSTB2 Fusion Protein--
The
PstB2V5His6p expressed either in yeast or in Sf9
cells was detected by SDS-polyacrylamide gel electrophoresis and
immunoblotting using the mouse monoclonal IgG directed against the V5
epitope. For yeast preparations, cytosols and membranes were isolated
as described earlier, in a buffer containing 50 mM
Tris-HCl, pH 8, 10 mM 2-mercaptoethanol, 0.3 M
sucrose, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. Sf9 cells were disrupted by probe sonication in an ice water bath for 15 s followed by a 30-s pause in ice water. The burst-pause cycle was repeated five times resulting in the suspension becoming translucent. This homogenate was subjected to ultracentrifugation at 100,000 × g for 1 h at
4 °C. The supernatant (cytosol) was collected, and the pellet
(membranes) was resuspended in the homogenization buffer. When yeast or
Sf9 membranes were examined for membrane binding of
PstB2V5His6p, the total membrane fractions were resuspended
in homogenization buffer containing 2 M KCl at 4 °C for
1 h to strip off peripheral membrane proteins and subsequently
centrifuged at 100,000 × g for 1 h. This washing step was repeated once before the resulting membrane pellet was resuspended in buffer without KCl. To test PSTB2V5His6p
binding, the KCl- washed membranes were mixed with the appropriate
yeast or Sf9 cytosols in a final volume of 200 µl of
homogenization buffer. The reaction mixtures were incubated at 30 °C
for 30 min, shifted to an ice bath, and finally subjected to
centrifugation at 100,000 × g. The resultant membrane
pellets were washed twice by resuspension in cold homogenization buffer
and recentrifuged. The final membrane pellets were resuspended in the
sample loading buffer for SDS-polyacrylamide gel electrophoresis,
immediately boiled, and then electrophoresed on an 8-16% Tris-glycine
slab gel. Proteins separated on the gel were transferred to
nitrocellulose membrane for immunoblot analysis. An anti-V5 monoclonal
antibody (Invitrogen) was used for detection of the fusion protein and a horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody
(Bio-Rad) was used as a secondary antibody. Color development of the
immunoblot used a diaminobenzamidine substrate. The nitrocellulose blots were blocked for 30 min with 5% skim milk and 1% Triton X-100
in PBS and then incubated with monoclonal antibody in 1% skim milk in
PBS buffer for 2 h. Unbound primary antibody was removed by
washing the blot three times with blocking buffer. Secondary antibody
was incubated with the blot for 1 h in blocking buffer followed by
two washes with 1% Triton X-100 in PBS. The blot was washed two
additional times in PBS and placed in a PBS solution containing 0.03%
hydrogen peroxide and 0.1% (w/v) daminobenzidine. Color development
was stopped by washing the blot with distilled water. In some
experiments immunoreactive material was detected by chemilumenescence
using a 100 mM Tris-HCl, pH 8.5, solution containing 225 µM courmaric acid, 1.25 mM luminol, and
0.003% hydrogen peroxide. The His6 epitope-tagged Sec14p
and PstB2p were detected in cytosolic fractions from E. coli
using high affinity anti-His6 antibody
(CLONTECH). The cytosolic fractions were subjected to electrophoresis and ligand blotting as described above except that
the anti-His6 antibody was used as the primary antibody.
Subcellular Fractionation of the Labeled Permeabilized
Cells--
Permeabilized yeast cells were prepared as described
previously (32). [3H]serine was incorporated into the
permeabilized cells in a reaction volume of 3 ml including 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 -chloro-alanine, 0.2 mM EDTA, 0.4 mM MgCTP, 0.28 M sorbitol, 0.27 M
mannitol, 50 µM L-serine, 100 µCi of
[3H]serine, and the permeabilized cells equivalent to 2.7 mg of protein. The reaction mixture was incubated at 30 °C for 100 min and then immediately homogenized gently on ice with 15 strokes using the B pestle of a Dounce homogenizer. Cell debris was removed from the homogenate by centrifugation at 1500 × g for
5 min. The supernatant (S1.5) was further centrifuged at 30,000 × g for 15 min to remove dense membranes. The supernatant
(S30) was removed from the pellet (P30), overlaid on a two step
gradient consisting of 1 ml of 80% sorbitol and 1 ml of 25% sorbitol,
and centrifuged for 2 h at 280,000 × g in a
Beckman SW41 rotor. All sorbitol densities are given as the w/v in 10 mM triethanolamine, pH 7.2. The interface between the 25 and 80% sorbitol layers was collected and adjusted to 43% sorbitol
using refractometry and layered on a gradient prepared in 40, 43, 60, 70, and 80% increments of 2.3 ml each. Membranes were separated by
centrifuging the gradient in an SW41 rotor at 280,000 × g for 40 h. Fractions were collected by aspiration from
the top of the gradient and stored at 20 °C.
Analysis of Subcellular Fractions--
Fractions obtained from
the sorbitol gradients were subjected to lipid and enzyme analysis and
antigen detection. PtdSer synthase and decarboxylase were measured as
described above. The vacuolar marker H+-ATPase was detected
by enzyme-linked immunosorbant assays (33). The late Golgi marker,
Kex2p protease, was determined by using Boc-Gln-Arg-Arg-7 amidomethyl
coumarin as the substrate (34). Lipids were extracted from the
fractions using the method of Bligh and Dyer (35) and further analyzed
by thin layer chromatography as described above.
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RESULTS |
Isolation and Characterization of the pstB2 Mutant--
Strains
containing a psd1::TRP1 allele must synthesize
virtually all of their PtdEtn by transporting nascent PtdSer to the locus of the Psd2p in the Golgi/vacuole, when grown in the absence of
Etn (see Fig. 1). Mutagenesis of these
strains can yield new strains that are Etn auxotrophs with mutations in
steps between PtdSer synthesis and decarboxylation that are likely to
be defective in lipid transport. We mutagenized the strain RYY52
(MAT lys2 trp1 ura3 his3 leu2 suc2
psd1-1::TRP1) and screened 150,000 survivors for the
presence of Etn auxotrophs. We obtained 32 new Etn auxotrophs, 9 of
which were psd2 mutants, 2 of which were pss
mutants, and 23 of which belonged to a new category. Outcrossing and
complementation analysis revealed that three strains belonging to a
single complementation group had a strong growth phenotype and some of
the biochemical properties expected for putative pstB
mutations. One of these strains was designated pstB2 and
further characterized.
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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. In addition, Etn and choline (Cho) can
be incorporated into PtdEtn and PtdCho at the endoplasmic reticulum via
CDP choline and CDP ethanolamine intermediates. The latter
pathways bypass the requirements for interorganelle PtdSer and
PtdEtn transport for the formation of PtdEtn and PtdCho,
respectively. Both known and proposed mutations in the metabolic and
transport process are shown in lowercase italic: pstA,
PtdSer transport A pathway; pstB, PtdSer transport B
pathway; 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.
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The growth properties of the pstB2 mutant were studied and
the findings are shown in Fig.
2A. The parental strain and
the pstB2 strain exhibit similar growth kinetics when
maintained on minimal medium containing Etn. In contrast, removal of
Etn from the medium has little effect upon the parental strain but is
lethal for the pstB2 strain. The pstB2 strain
appears to divide twice before growth arrest. After prolonged
incubation the cells lyse. These results demonstrate that the
pstB2 strain is an Etn auxotroph and that the growth
phenotype observed in solid medium is also apparent in liquid
medium.
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Fig. 2.
The pstB2 strain is an Etn
auxotroph with defective lipid metabolism. A, the
Etn growth phenotype. Both the parental strain
(psd1, circles) and the pstB2
strain (psd1/pstB2, triangles) were grown in
SC medium plus Etn at 30 °C to mid-log phase. Cultures were then
washed once and diluted to an A600 nm of 0.02 in
SC medium either with (solid symbols) or without
(open symbols) Etn. The diluted cultures were incubated at
30 °C, and the A600 nm of each culture was
monitored at the indicated time points throughout the experiment. The
results represent the average of three independent experiments
performed in duplicate. B, aminoglycerophospholipid
metabolism of the pstB2 strain. The
aminophospholipid composition of the pstB2 strain was
determined by following the incorporation of
L-[3H]serine as described under
"Experimental Procedures." Data are expressed as the percentage of
label in individual phospholipid over total radiolabel in each strain.
PtdOH, phosphatidic acid. Other abbreviations are the same
as Fig. 1. The results are the average of four independent experiments
performed in duplicate. Values are mean ± S.E.
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The pstB2 strain and its parental strain were examined for
defects in lipid metabolism by following the incorporation of
[3H]serine into aminoglycerophospholipids and the results
are shown in Fig. 2B. In parental cells,
[3H]serine is readily incorporated into PtdSer, PtdEtn,
and PtdCho. In the pstB2 strain, [3H]serine is
also incorporated into all three lipids, but the pattern of labeling is
markedly altered. Relative to the parental strain, the mutant shows a
50% increase in the labeling of the PtdSer pool and a 72% decrease in
the labeling of the PtdEtn pool. This finding is consistent with a
defect in the formation of PtdEtn from PtdSer in the mutant. There is
also a modest change in the labeling of the PtdCho pool, but analysis
of this lipid pool is not straightforward because
[3H]serine can significantly label this lipid via the one
carbon pathway (8, 12).
A trivial explanation of the reduced PtdEtn formation in the
pstB2 strain is that it is a consequence of defective Psd2p. However, genetic experiments in which the pstB2 strain is
crossed with a psd1::TRP1
psd2::HIS3 double mutant reveal that the activity of
Psd2p within the pstB2 strain is sufficient to support
normal growth in the absence of Etn. Direct biochemical measurement of Psd2p catalytic function further demonstrates that the enzyme activity
in the mutant is 75% of that found in the parental strain (Fig.
3). In addition, increased expression of
Psd2p by transformation of pstB2 mutant strains with a
centromeric plasmid harboring the PSD2 gene does not rescue
the mutants. These findings make it unlikely that a mutation in the
structural gene for PSD2 is responsible for the alteration
in lipid metabolism. The biochemical phenotype of the pstB2
mutant is also inconsistent with a defect in PtdSer synthase, as
genetic crosses demonstrate that the pstB2 and
pss strains can complement each other. The PtdSer synthase
activity of the pstB2 mutant is also normal (120% relative
to parental strain). We also used genetic manipulation of
PSD1 to investigate whether the defect in pstB2
was directly linked to PtdSer metabolism. If the defect in the
pstB2 mutant is genuinely coupled to the cell's need for
PtdEtn, then provision of this lipid by the action of Psd1p, instead of
exogenous Etn, should bypass the mutation. Transformation of the
pstB2 strain with a plasmid harboring the PSD1
gene effects complete bypass of the pstB2 mutation.
Collectively, the characteristics of the pstB2 mutant
described above are those expected for strains with defects in the
transport of nascent PtdSer to the locus of Psd2p.
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Fig. 3.
PSD2 activity of the pstB2
strain in the absence or presence of plasmids harboring wild type
PSD2 or PSTB2 genes. All strains
were harvested at mid-log phase in SC+Etn medium with or without uracil
at 30 °C. Cell-free extracts were prepared, and PSD2 activities were
measured as described under "Experimental Procedures."
PSD2 activities are presented as the percentages relative to the
activity of parental control (100%). The results represent at least
four independent experiments performed in duplicate. Values are
mean ± S.E.
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Cloning and Characterization of the PSTB2 Gene--
The strong
growth phenotype of the pstB2 mutant in the context of a
psd1 mutation enabled the cloning of a complementing gene. The pstB2 strain was transformed to Ura+
prototrophy using a YCp50 vector harboring a yeast genomic library. ~1.3 × 105 Ura+ transformants were
further examined for Etn prototrophy by replica plating onto minimal
medium lacking both Ura and Etn. We identified 63 strains that
displayed the ability to grow in the absence of both Ura and Etn. From
the pool of 63 transformants, we identified 53 that coordinately lost
both Ura and Etn prototrophies after growth for 30 generations in
nonselective medium. These plasmid loss experiments demonstrated
genetic linkage between vector-encoded and genomic insert-encoded
functions. Five yeast strains containing the plasmid that conferred Etn
prototrophy were randomly selected, and the plasmid was recovered by
shuttling into E. coli. (22). Reintroduction of these
plasmids into the pstB2 mutant restored the growth to wild
type levels in the absence of Etn. Restriction endonuclease analysis
revealed that all five of the strains contained a plasmid with
identical or partially overlapping genomic inserts.
The recovered plasmid was subjected to DNA sequence analysis that
revealed the nucleotide sequence at both ends of the genomic insert.
The genomic insert was identified as a 10-kb pair piece of DNA derived
from chromosome XIV that included 7 complete and 2 partial ORFs. Each
of the ORFs was subcloned and examined for complementation of the
growth defect of the pstB2 mutant. Only one of the ORFs,
designated PDR17 in the S. cerevisiae Genomic Data base, complemented the mutant strain.
The PSTB2/PDR17 gene has recently been reported by van den
Hazel et al. (36) to be involved in hypersensitivity to
multiple drugs when its closest homologue, PDR16 gene, is
absent. Although the function of PSTB2/PDR17 in multiple
drug resistance of yeast was not elucidated in that report, they
demonstrated that a pdr17::HIS3 containing a wild
type copy of PSD1 in its genome has a moderate accumulation
of PtdSer and a decrement of PtdEtn levels. This is consistent with our
result that PSTB2/PDR17 plays a role in PtdSer metabolism.
The characteristics of the pstB2 strain grown without Etn
either in the presence or the absence of the plasmid harboring the PSTB2/PDR17 gene are shown in Fig.
4A. The PSTB2/PDR17
gene restores the growth of the mutant strain to levels that are
equivalent to that of the parental strain. The ability of the genomic
insert to correct the biochemical phenotype of the mutant is shown in Fig. 4B. Both the accumulation of PtdSer and the markedly
reduced levels of PtdEtn found in the pstB2 strain are
rectified by the PSTB2 gene present on either low copy
(YCp50) or high copy (YEp352) plasmids. A convenient way to compare
strains with defects in aminoglycerophospholipid metabolism is to
express the activity as a ratio of product (PtdEtn) to precursor
(PtdSer) as shown in Fig. 4C. In parental strains the
PtdEtn/PtdSer ratio is 0.44, whereas in the mutant strain the value is
0.07. In contrast, mutant strains with the PSTB2/PDR17 gene
present on high copy or low copy plasmids have PtdEtn/PtdSer ratios
that are essentially the same as that of the parental strain. These
findings establish that the PSTB2/PDR17 gene complements the
biochemical as well as the growth defect of the pstB2
mutant.
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Fig. 4.
The wild type PSTB2/PDR17
gene restores both Etn prototrophy and the aminophospholipid
composition of the pstB2 strain. A,
the Etn-independent growth of the parental (open circles)
and of the pstB2 strain either with (solid
squares) or without (open triangles)
YEp352-PSTB2/PDR17 plasmid. All growth measurements were
performed as described in Fig. 2, except the strain carrying the
YEp352-PSTB2/PDR17 plasmid was grown in SC medium lacking
uracil. B, lipid labeling and composition. Data are
expressed as the percentage of radiolabel present in the phospholipid
classes for each strain. The YCp50 and YEp352 plasmids are low and high
copy, respec- tively. C, ratio of PtdEtn to PtdSer in parental and
pstB2 strains in the absence or presence of plasmids with
the complementing gene. Results in all panels represent the averages of
two to five independent experiments performed in duplicate.
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The effects of PSTB2/PDR17 gene expression on Psd2p activity
are shown in Fig. 3. Transformation of the pstB2 strain with the wild type gene has no significant effect upon PtdSer decarboxylase activity measured in cell extracts. These findings suggest that PstB2p
does not directly modulate PtdSer decarboxylase activity. We have also
constructed null alleles of the PSTB2/PDR17 gene using the
single step disruption procedure of Rothstein (24). The construct used
for the disruption is shown in Fig.
5A. Verification of the
integration site of the construct was determined by PCR using primers
that only recognize the disrupted gene present in the correct locus.
The results shown in Fig. 5B demonstrate the presence of the
null and wild type alleles in two different diploid strains. The
diploid strains were induced to sporulate, and the resultant tetrads
were dissected. Of 20 tetrads analyzed, all showed 2:2 segregation of
His+ and His phenotypes. All the
His+ strains from the wild type diploid (SEYd) are viable
and have no apparent growth defect. This result agrees with the
previous report that the PSTB2/PDR17 gene is not
essential (36). However, all His+ strains from diploid
WWYd9 carrying double psd1 null alleles were Etn
auxotrophs, demonstrating the synthetic lethality between pstB2::HIS3 and psd1. The
PtdEtn/PtdSer ratio of strains containing the
pstB2::HIS3 allele is shown in Fig. 4B
along with that of the parental and the pstB2 mutant
strains. The pstB2::HIS3 allele reproduces the
growth and biochemical phenotype of the pstB2 mutant derived
by mutagenesis. The results strongly suggest that PstB2/Pdr17p function
is biochemically linked to Psd2p-dependent metabolism of
PtdSer.
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Fig. 5.
Disruption of the PSTB2/PDR17
gene. A, schematic of the disruption of
PSTB2. The PSTB2 gene was disrupted by inserting
a BamHI DNA fragment containing the HIS3 gene
into the coding sequence of PSTB2 at the BclI
site. The linear pstB2::HIS3 DNA used for
replacing the genomic PSTB2 gene was excised from a
pGEM4Z-pstB2::HIS3 construct by SacI
and XbaI digestion. The SacI site was derived
from the YEp352 shuttle vector. Lightly shaded area, genomic
DNA outside the PSTB2 coding region that is included in the
linear pstB2::HIS3 construct. Open
area, genomic DNA sequence of chromosome XIV adjacent to the
region used to replace wild type PSTB2. The large
arrows indicate the direction of transcription. B,
confirmation of genomic PSTB2 disruption by PCR. Both
PSD1/his3 and psd1/his3 diploid strains were
transformed with the linear construct of
pstB2::HIS3 shown in A. Three
His+ transformants of each diploid strain were randomly
selected for identifying genomic pstB2::HIS3 by
PCR reaction using the three primers illustrated by the small
arrows in A. The presence of a wild type allele is
indicated by a 1.9-kb fragment, and the disrupted allele is shown by a
1.5-kb fragment. Lanes 1 and 11, 1kb ladder;
lanes 2-4, transformed wild type diploid; lane
5-7, transformed psd1::TRP1 diploid;
lane 8, wild type haploid; lane 9, pstB2::HIS3 plasmid; lane 10, wild type
PSTB2 plasmid.
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Sequence analysis of the PSTB2/PDR17 gene establishes that
it is related to the PtdIns/PtdCho transfer protein encoded by the
SEC14 gene. The PSTB2/PDR17 has also been
reported as a member (SFH4) of five yeast
Sec14p homologues (SFHs)
(37). The sequence alignment of the coding regions of the genes is
shown in Fig. 6. The deduced protein
sequences demonstrate a central region of homology between Sec14p and
PstB2/Pdr17p comprising ~27 kDa. The sequence similarity in the
central region is 58%. In contrast the amino- and carboxyl-terminal
regions of the two proteins are markedly divergent with little
similarity. The nonhomologous amino-terminal region of PstB2p is 11 kDa
in size, and the carboxyl-terminal region is 3 kDa in size.
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Fig. 6.
Alignment between PstB2/Pdr17p and
Sec14p. The amino acid sequences of PstB2/Pdr17p and Sec14p were
aligned using the pair wise sequence alignment services under the
Baylor College of Medicine search launcher. Identical amino acids are
indicated by a colon, whereas similar amino acids are
indicated by a dot. The overall identity and similarity
between PstB2/Pdr17p and Sec14p are 22.4 and 49.4%, respectively. The
central region of PstB2/Pdr17p, which has the highest similarity to
Sec14p (57.5%), is underlined.
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Properties of the PstB2/Pdr17p--
The discovery that the
PstB2/Pdr17p is related to the Sec14p raises the question of whether
the protein functions as a lipid transfer protein, perhaps specific for
PtdSer. To examine this question we expressed the PstB2p using a
baculovirus vector and Sf9 cells. For these studies we employed
both normal and carboxyl-V5His6 epitope-tagged
PstB2/Pdr17p. From genetic studies with constructs encoding the
PstB2/Pdr17-V5His6p, we know that this form of the protein
restores the growth of pstB2 mutants (data not shown). Overexpression of PstB2/Pdr17-V5His6p in yeast and
Sf9 cells yields only weak phospholipid transfer activity
detectable above the normal background activity of Sf9 cells. In
contrast, expression a His6-PstB2/Pdr17p in E. coli provides a source of crude protein with significant transfer
activity for PtdIns as shown in Fig. 7.
Comparison of immunoblots that detect the epitope tag on PstB2p expressed in bacteria and Sf9 cells surprisingly demonstrates that the latter contain ~10 times the amount of the recombinant protein. We also compared the level of Sec14p expression to that of
PstB2p in E. coli. The Sec14p was expressed at ~25 times
the level of PstB2p. Thus the specific transfer activity of recombinant PstB2p appears to be nearly five times greater than that for Sec14p. We
also investigated the activity of the E. coli-derived
protein with PtdSer as a substrate and were unable to detect any
transfer of this lipid. Thus, the recombinant PstB2/Pdr17p clearly has in vitro activity related to the Sec14p. We next examined
the reciprocal complementation of sec14ts and
pstB2 strains by their wild type genes. Overexpression of the PSTB2/PDR17 gene can complement the
sec14ts growth defect. In contrast
overexpression of the SEC14 gene fails to rescue the
pstB2 mutant.
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Fig. 7.
The PstB2/Pdr17p exhibits PtdIns but not
PtdSer transfer activity in vitro. PtdIns and
PtdSer transfer activities of His6-tagged-PstB2/Pdr17p and
Sec14p were measured in extracts from E. coli overexpressing
either protein. The transfer activities were expressed as the
percentage of [3H]PtdIns or [3H]PtdSer
transferred over total [3H]PtdIns- or
[3H]PtdSer-containing rat liver microsomes used in the
assays. All cell extracts were matched for a protein content of 75 µg/assay. Ctrl, E. coli without plasmid; Blank,
buffer control. The result is an average of two independent experiments
performed in duplicate.
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Examination of the subcellular distribution of PstB2/Pdr17p reveals
that it is both soluble and membrane bound as shown in Fig.
8A. Part of the membrane-bound
population can be removed by 2 M KCl washing of the
membranes (Fig. 8A). However, a significant portion of the
membrane bound population remains resistant to removal by high salt
washing. Soluble preparations of the epitope-tagged PstB2/Pdr17p
derived from recombinant baculovirus-infected Sf9 cells were
used to examine the reversibility of the membrane association. The
results shown in Fig. 8B demonstrate that the soluble form of PstB2/Pdr17p will readily associate with yeast microsomal membranes. In additional experiments (data not shown) we verified that the cytosolic form of the protein did not nonspecifically precipitate under
the conditions used for membrane binding. The nature of the membrane
association is currently not understood. However, liposome binding
experiments using membranes composed of PtdCho alone or in binary or
ternary mixtures with PtdSer, PtdIns, phosphatidylinositol 4-phosphate,
and phosphatidylinositol 4,5-bisphosphate do not reveal any high
affinity interactions (data not shown). The results suggest that the
association of PstB2/Pdr17p with membranes may be mediated by resident
proteins.
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Fig. 8.
The PstB2/Pdr17p is amphitropic.
A, expression and cellular distribution of PstB2/Pdr17p.
V5His6 epitope-tagged PstB2/Pdr17p was expressed in a
pstB2 strain grown in minimal medium containing galactose
and raffinose as the carbon sources. Cell-free extract (CE),
cytosol (Cyt), membranes before KCl wash (M), KCl
washed membranes (M*), and KCl washing supernatant
(W) were prepared as described under "Experimental
Procedures." All fractions (2.3 µg of protein) were subjected to
SDS-polyacrylamide gel electrophoresis and immunoblot analysis.
Epitope-tagged PstB2/Pdr17p was detected using mouse anti-V5 epitope
monoclonal antibody and visualized by colorimetric reaction of
horseradish peroxidase. MW, molecular mass standards.
B, association of cytosolic PstB2/Pdr17p with yeast
microsomes. Sf9 cytosol (10 µg of protein) from cells
expressing the epitope-tagged PstB2/Pdr17p was mixed with 22 µg of
protein of KCl washed yeast microsomes from wild type PSTB2
yeast as described under "Experimental Procedures." In lanes
a and b the 100,000 × g pellet was
examined and in lane d the 100,000 × g
supernatant was examined for the presence of the V5 antigen. Sf9
cells expressing yeast Dpp1p were used as an Sf9 infected
control.
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Lipid Accumulation in pstB2 Mutants Occurs in the Golgi and a Light
Membrane Fraction--
When examined at the whole cell level, the
pstB2 mutant strain exhibits a relative accumulation of
PtdSer and reduced formation of PtdEtn (Figs. 2B and 4,
B and C). We examined subcellular fractions derived from both mutant and parental strains to determine where in the
cell the PtdSer accumulates. For these experiments we conducted labeling studies and performed subcellular fractionation on
permeabilized cells. We have established that permeabilized cells
faithfully recapitulate the lipid transport processes of wild type
cells and the lipid accumulation defect of mutant cells. The detailed properties of the permeabilized cells will be described in another publication. We have found it necessary to use permeabilized yeast cells because we are unable to obtain good separation of organelles from intact cells when they are grown on minimal medium. In addition, our procedure (and most others) used spheroplasts as the starting point
for isolation of organelles. The time required for spheroplasting cells
at the end of a labeling experiment adds a confounding and unwanted
variable into the experiments that is eliminated by using permeabilized
cells. The results in Fig. 9,
A and B, show the distribution of PtdSer
synthase, Kex2p protease, and vacuolar ATPase using our procedure. By
these criteria the resolution of elements of the endoplasmic reticulum
specific for PtdSer synthesis, the Golgi apparatus, and the vacuolar
compartment is exceptional. The Psd2p, which metabolizes PtdSer to
PtdEtn, is found in both the Golgi and vacuolar compartments (Fig.
9C). The amount of Psd2p found in the Golgi compartment from
permeabilized cells is low but significant.
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Fig. 9.
Subcellular fractionation of permeabilized
yeast. A, sedimentation of the endoplasmic reticulum by
30,000 × g centrifugation. The PSS activity was used
as the endoplasmic reticulum marker. PSS activity is expressed as the
percentage of the enzyme activity in the 30,000 × g
supernatant (S30) or pellet (P30) over the combined activities from
both fractions. The data are mean ± S.E. from three independent
experiments. B, separation of the Golgi and the vacuole
compartments. The Golgi and the vacuolar membranes resolved on sorbitol
gradients were identified by following the Golgi marker, Kex2p
peptidase activity, and vacuolar H+-ATPase reactivity to
the antibodies against either the 60- or the 100-kDa subunit of
vacuolar H+-ATPase. C, distribution of PSD2
activity on sorbitol gradients. The PSD2 activity of each fraction of
the sorbitol gradient was expressed as the percentage the total PSD2
activity applied on the gradient. The data shown in B and
C are representative results from at least four separate
gradients for each strain.
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We examined the subcellular distribution of nascent PtdSer and PtdEtn
by labeling permeabilized cells with [3H]serine and
subsequently using the gradient analysis procedures depicted in Fig.
10. The data presented in Fig. 10
reveal several important details about lipid translocation and
metabolism in both wild type and mutant yeast strains. In wild type
strains clear evidence is presented demonstrating nascent PtdSer
and PtdEtn in the Golgi compartment and a novel light
membrane fraction. Strikingly, there is almost no newly
synthesized PtdSer or PtdEtn in the vacuolar compartment.
Paradoxically, the vacuolar compartment as shown in Fig. 9 contains the
majority of Psd2p. This finding suggests that in the permeabilized
cells there is little PtdSer transport to and decarboxylation by the
vacuolar compartment. The findings with the wild type cells are
consistent with the Golgi apparatus being the principal site of PtdSer
decarboxylation in this system. Very significant labeling of PtdSer and
PtdEtn is also observed in a light membrane fraction that is located at
the top of the sorbitol gradient. This membrane fraction does not
contain PtdSer synthase, Kex2p, or vacuolar H+-ATPase
activity. The origin of the light membrane fraction is currently being
investigated.
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Fig. 10.
The pstB2 strain accumulates
PtdSer in the Golgi and a light membrane fraction. Permeabilized
wild type (psd1/PSTB2, A) and pstB2
(psd1/pstB2, B) cells were labeled with
[3H]serine and then subjected to subcellular
fractionation as described under "Experimental Procedures." The
gradient in A was loaded with interface membranes containing
2.3 × 105 cpm lipid. The gradient in B was
loaded with interface membranes containing 1.5 × 105
cpm lipid. LM indicates the location of the light membrane
fraction. The result is a representative one of three separate
gradients for each strain. Open circle, PtdSer; solid
circle, PtdEtn.
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Comparison of the pstB2 strain with the wild type strain
demonstrates important differences in the lipid-labeling
patterns. The mutant strain accumulates PtdSer relative to
PtdEtn in the Golgi and light membrane fractions. The ratio of
PtdEtn/PtdSer is 0.3 for the Golgi and 0.3 for the light membranes of
wild type cells but less than 0.04 for the corresponding fractions from the mutant cells. The results are consistent with PstB2/Pdr17p playing
an integral role in controlling the access of nascent PtdSer to Psd2p.
Control experiments establish that the PstB2/Pdr17p does not act as a
cofactor for Psd2p or enhance its activity in vitro. The
results from the [3H]Ser labeling experiments also
clearly demonstrate that the pstB2 mutation does not prevent
the appearance of PtdSer in either the Golgi or light membranes. It is
possible that PstB2/Pdr17p affects the appearance and presentation of
PtdSer to a specific subcompartment of the Golgi.
We next sought to determine the distribution of PstB2p in the
subcellular fractions derived from the permeabilized cells. Constructs
expressing PstB2p with a V5His6 epitope tag were used for
localization. Genetic experiments demonstrate that the epitope-tagged version of PstB2/Pdr17p complements the null allele of the
PSTB2/PDR17 gene. Fig.
11A is an immunoblot
analysis of equivalent quantities (adjusted for volume differences) of
the major fractions derived from the permeabilized cells. The
PstB2/Pdr17p found in the cell homogenate resides in the S30 and P30
fractions. Only minor amounts of PstB2/Pdr17p are recovered in the
interface fraction (used for the isolation of Golgi and vacuole
membranes) that is derived from the S30 fraction. More detailed
analysis of the distribution of PstB2/Pdr17p in the Golgi and vacuole
fractions is shown in Fig. 11B. The membranes were recovered
from these latter fractions by 10-fold dilution of the sorbitol and
sedimentation at 280,000 × g. The PstB2/Pdr17p is
found associated with light membranes, the Golgi, and vacuole fraction.
A significant proportion of the PstB2/Pdr17p found in the denser
vacuole fraction shows evidence of proteolytic degradation. These
findings indicate that the subcellular distribution of PstB2/Pdr17p is
broad and not restricted to either the cytosol or a specific organelle
membrane.
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Fig. 11.
Membrane association of PstB2p is not
organelle specific. A yeast strain (psd1/pstB2)
containing a complementing pYES-PSTB2-V5His6
plasmid was permeabilized and subjected to subcellular fractionation as
described under "Experimental Procedures." A, an
identical percentage of the homogenate (H), S30, P30, and
interface (I) fractions was electrophoresed and subjected to
immunoblot analysis. The epitope tag was detected with mouse anti-V5
monoclonal antibody. B, the interface fraction was resolved
on sorbitol gradients as described in Fig. 10 and under "Experimental
Procedures." The membranes in the gradient fractions were recovered
by 10-fold dilution of the sorbitol and centrifugation at 280,000 × g for 2 h. The membrane pellets were harvested
directly in gel loading buffer, electrophoresed, and subjected to
immunoblot analysis as described above. LM indicates the
location of the light membrane fraction.
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DISCUSSION |
The mechanisms by which phospholipids are transported from their
sites of synthesis to the multiplicity of organelles found in
eukaryotic cells are poorly understood. We have approached this problem
by devising a genetic screen, outlined in Fig. 1, to identify new yeast
strains defective in the transport-dependent metabolism of
PtdSer. A new strain, pstB2, was identified; its complementing gene, PSTB2/PDR17, was cloned, partially
sequenced, and mapped, and the corresponding null allele was created
.
Both the original pstB2 mutant and strains engineered to
contain pstB2/pdr17::HIS3 null alleles exhibit the
same growth characteristics, consisting of conditional synthetic
lethality with psd1 mutations and markedly reduced formation
of PtdEtn from a PtdSer precursor. The defect in PtdEtn formation is
related to the transport-dependent metabolism of PtdSer. In
the psd1 genetic background, PtdSer must be transported
to Golgi and vacuolar compartments for decarboxylation. Examination of
aminoglycerophospholipid metabolism at the whole cell level clearly
shows an accumulation of the precursor, PtdSer, and a significant
decrement in the formation of PtdEtn. Genetic and enzymatic analysis
eliminate specific lesions in Psd2p function as a cause of the
reduction in PtdEtn formation. In addition, we can not find any
evidence for mislocalization of Psd2p (Fig. 9C). Consistent
with the latter findings is the normal localization of Kex2p and the
vacuolar H+-ATPase (Fig. 9B), indicating that
the pstB2 lesion does not cause general protein sorting defects.
The growth phenotype and the abnormal lipid metabolism of the
pstB2 strains are complemented by a gene we name
PSTB2. This gene has recently been reported as
PDR17, which causes hypersensitivity of yeast to multiple
drugs when deleted from the genome along with its closest homologue,
PDR16 (36). However, deletion of the PSTB2/PDR17
gene alone did not increase drug sensitivity (36), and this strain
showed perturbations in lipid metabolism much milder than the
pstB2 mutant in a psd1 genetic background.
Because the synthetic lethality between pstB2/pdr17 and
psd1 suggests that PSTB2/PDR17 gene is
involved in the Psd2p-dependent PtdSer metabolism, it is
likely that the wild type allele of PSD1 in the
pstB2::HIS3/pdr17 cell provides adequate
amounts of PtdEtn to maintain membrane permeability as well as viability.
The PSTB2/PDR17 gene is structurally related to the
SEC14 gene encoding a PtdIns/PtdCho transfer protein. The
deduced amino acid sequences for PstB2/Pdr17p and Sec14p consist of a
central core region of 27 kDa and 58% homology. Relative to Sec14p,
PstB2/Pdr17p has a divergent amino-terminal region of 11 kDa and a
divergent carboxyl-terminal region of 3 kDa. The PstB2/Pdr17p is
amphitropic and is found in both cytosolic and total membrane fractions
of yeast cells. Similar amphitropic properties have been described for
Sec14p (38). There does not appear to be any specificity in the
membrane association of PstB2/Pdr17p. Recombinant forms of the
PstB2/Pdr17p produced in Sf9 cells also exhibit the amphitropic character, and the soluble form will readily bind to yeast microsomal membranes. The structural similarity between Sec14p and PstB2/Pdr17p raises the question of whether the latter has lipid transfer activity. In the original reports of lipid transfer activity in cytosol from
sec14ts mutants, almost no activity in addition
to that ascribable to Sec14p could be detected. We also find that lipid
transfer activity attributable to PstB2/Pdr17p is minimal in yeast
cells or Sf9 cells overexpressing the protein. At present, we do
not know if our inability to measure phospholipid transfer effected by
PstB2p derived from eukaryotes is because of the presence of inhibitory factors or post-translational modification. It is unlikely that the
V5His6 epitope tag renders the protein inactive, because
this structural variant effectively rescues strains with pstB2
alleles. However, overexpression of PstB2/Pdr17p in E. coli yields protein preparations in which PtdIns transfer activity
is easily measured. Thus, it is clear that PstB2/Pdr17p has intrinsic
lipid transfer activity in vitro. The transfer activity is
presumably related to its function in vivo, but it remains
unclear as to whether this is a true lipid transfer function for the
purpose of membrane biogenesis via a soluble carrier within the cell.
Direct tests of PstB2/Pdr17p as a cofactor or stimulator of Psd2p
demonstrate that it does not enhance the catalytic activity of the
PtdSer decarboxylase in vitro. High level expression of the
PSTB2/PDR17 gene can also function to suppress
sec14 mutations. This surprising result implicates
PstB2/Pdr17p in some aspects of Golgi function.
We have further established that the defect in the transport dependent
metabolism of PtdSer observed at the whole cell level is also seen in
permeabilized cells. The fidelity of the permeabilized cell system to
the intact cell system, with respect to lipid transport has now
provided the means to assess the location of PtdSer accumulation in
mutant cells relative to their wild type counterparts. Previously, this
has been a difficult problem to overcome insofar as the subcellular fractionation schemes typically used to isolate Golgi, vacuoles, and
plasma membrane from cells grown on rich medium have not been successful with cells grown on synthetic medium. Using permeabilized yeast cells as the starting point for subcellular fractionation we
demonstrate that we can resolve the Golgi, vacuoles, endoplasmic reticulum, and a novel light membrane fraction extremely well. Examination of the distribution of newly synthesized
aminoglycerophospholipids among the subcellular fractions provides
significant new information about the site of action of PstB2/Pdr17p
and Psd2p. The results presented in Fig. 10 demonstrate that
significant amounts of the decarboxylated PtdSer reside within the
Golgi apparatus and a light membrane fraction of permeabilized cells.
Surprisingly, little detectable decarboxylation of PtdSer occurs in the
vacuolar compartment, even though this is where the majority of the
decarboxylase is located. The results suggest that most of the
decarboxylation in the permeabilized cells occurs in the Golgi.
However, we can not completely rule out that a rapidly transported pool
of PtdSer is decarboxylated elsewhere.
The PstB2/Pdr17p appears to play a critical function in regulating the
decarboxylation of PtdSer. Precisely how PstB2/Pdr17p functions is not
yet known. The data clearly indicate that the protein binds membranes
in vitro and has an affinity for PtdIns as measured by the
lipid transfer assay. However, based upon our liposome binding studies,
the high affinity binding of the protein to membranes requires
components other than phospholipids. The role of the membrane binding
in PstB2p function is currently unknown. However, previous studies (9)
have identified Stt4p as another protein involved in the pstB pathway.
It is feasible that PstB2p could bind and modulate the activity of Stt4p.
Empirically our data support a role for PstB2/Pdr17p in regulating the
access of PtdSer to the decarboxylase. It is noteworthy that the
pstB2 mutation does not prevent the transport of PtdSer to
the Golgi or the appearance of PtdSer in the light membrane fraction
(see Fig. 10). The nature of the light membrane fraction is also not
known. The light membranes are of very low density as they do not
sediment in 40% sorbitol at 150,000 × g. Possible candidates for these membranes include retrograde vesicles, intra-Golgi vesicles, or anterograde vesicles exiting the trans Golgi. We currently
have a bias that these are retrograde vesicles, because PtdEtn formed
by the action of Psd2p must be transported to the endoplasmic reticulum
to synthesize PtdCho.
In summary, this report provides evidence that PstB2/Pdr17p is involved
in the Psd2p-dependent PtdSer metabolism to PtdEtn. The
similarity between PstB2/Pdr17p and Sec14p suggests that PstB2/Pdr17p may play a role in regulating PtdSer transport vesicles. The
amphitropic nature of PstB2/Pdr17p may be part of the regulatory
mechanisms to control its function.
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ABSTRACT
INTRODUCTION
