(Received for publication, November 8, 1994)
From the
Phosphatidylserine decarboxylase (PSD1) plays a central role in
the biosynthesis of aminophospholipids in both prokaryotes and
eukaryotes by catalyzing the synthesis of phosphatidylethanolamine.
Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R.(1993) J. Biol. Chem.268, 21416-21424; Clancey, C.
J., Chang, S.-C., and Dowhan, W.(1993) J. Biol. Chem. 268,
24580-24590) described the cloning of a yeast structural gene for
this enzyme (PSD1) and the creation of the null allele. Based
on the phenotype of strains containing a null allele for PSD1 (psd1-1::TRP1) it was hypothesized that yeast have a
second phosphatidylserine decarboxylase. The present studies
demonstrate the presence of a second enzyme activity (denoted PSD2)
which, depending on the method of evaluation, accounts for 4-12%
of the total cellular phosphatidylserine decarboxylase activity found
in wild type. Recessive mutations resulting in loss of this enzyme
activity (denoted psd2) in cells containing the psd1-
1::TRP1 null allele also result in ethanolamine
auxotrophy. When incubated with [
H]serine these
double mutants accumulate label in phosphatidylserine, while very
little (<5%) is converted to phosphatidylethanolamine. In addition,
these mutants have a
70% decrease in the amount of total
phosphatidylethanolamine even when grown in the presence of exogenous
ethanolamine. Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the
PSD2 enzyme activity. Unlike the PSD1 activity, the PSD2 enzyme
activity does not localize to the mitochondria, but to a low density
subcellular compartment with fractionation properties similar to both
vacuoles and Golgi.
Phosphatidylserine decarboxylase (PSD) ()is an
important enzyme in the synthesis of aminophospholipids. In Escherichia coli the decarboxylase functions in the only
pathway for phosphatidylethanolamine (PtdEtn)
synthesis(1, 2) . However, in eukaryotes, PtdEtn can
be synthesized by the action of the decarboxylase or ethanolamine
phosphotransferase(3, 4) . In addition, mammalian
cells possess a base exchange enzyme whose contribution to PtdEtn
synthesis in vivo is poorly understood(5) . Early
genetic examination of aminophospholipid synthesis by Atkinson et
al.(6) in the yeast Saccharomyces cerevisiae focused on the isolation of mutants requiring ethanolamine or
choline for growth. All of the mutants isolated belonged to a single
complementation group designated cho1 that had defects in the
enzyme phosphatidylserine synthetase. Interestingly, none of the
mutants were reported to have defects in PSD.
The auxotrophic requirement of the cho1 mutants is not only fulfilled by ethanolamine and choline, but also by monomethylethanolamine and dimethylethanolamine, indicating that phosphatidylcholine (PtdCho), rather than phosphatidylserine (PtdSer) or PtdEtn, is the essential end product(6, 7, 8) . Mutants having defects in either the first methylation step (pem1/cho2) or second and third methylation steps (pem2/opi3) in the conversion of PtdEtn to PtdCho do not require choline or ethanolamine for growth, presumably because low levels of PtdCho and/or the mono- and dimethyl forms of PtdEtn suffice. Strains with both pem1/cho2 and pem2/opi3 mutations, however, are stringently auxotrophic for choline, indicating that PtdEtn alone will not substitute for all the methylated phospholipids(8) .
Our initial efforts to isolate
yeast strains defective in PSD by isolating ethanolamine auxotrophs in
a wild type background failed entirely. ()Using alternative
methods, we were, however, able to isolate strains defective in PSD
activity, clone the gene encoding for the mitochondrial PSD (PSD1), and create yeast strains containing a psd1-
1::TRP1 gene disruption(9) . These null
mutants possessed <5% wild type PSD activity, yet did not require
ethanolamine for growth and had metabolic characteristics similar to
wild type cells. Similar findings were reported by Clancey et
al.(10) . These observations led us to hypothesize that
the yeast S. cerevisiae possesses two functional PSD
activities. The goals of the current studies were to: 1) describe the
presence of this second PSD activity (PSD2) in psd1-
1::TRP1 mutants, 2) isolate mutants lacking PSD2 activity (psd2)
by selecting ethanolamine auxotrophs in the psd1-
1::TRP1 background, and 3) identify the subcellular location of the PSD2
enzyme activity. The results unambiguously establish the presence of a
second, non-mitochondrial PSD activity. In addition, these studies
demonstrate that loss of both PSD1 and PSD2 enzyme activities results
in ethanolamine auxotrophy and impaired PtdSer turnover.
The growth media for yeast, YPD, minimal
medium plus methionine and auxotrophic requirements (SD), and minimal
medium containing all amino acids (SC), were prepared by standard
methods(14) , except where noted. Adenine and uracil (20
mg/liter) were routinely added to YPD to give YPDAU, and ethanolamine
was added where noted to give YPDAUE. Ethanolamine-supplemented media
contained 2-5 mM ethanolamine added from a 0.5 M, pH 5-6, sterile-filtered stock solution. Media for
subcellular localization experiments, except where noted, was that
described by Daum et al.(15) , referred to here as DBS
medium: 0.3% yeast extract, 1 g/liter each of glucose, KH PO
and NH
Cl, 0.5 g/liter each of
CaCl
2H
O and NaCl, 0.6 g/liter
MgSO
H
O, 0.3 ml/liter 1% FeCl
,
and 22 ml of 85% DL-lactic acid, adjusted to pH 5.5.
Cells exhibiting only the psd2 mutation (PTY22),
thereby expressing only PSD1 activity, were constructed from the psd1-1::TRP1, psd2 strain described above (PTY18) by
mating with the PSD1, PSD2, trp 1 strain (422). After
sporulation of the diploid, segregation of the tryptophan
(trp
) and ethanolamine (etn
)
auxotrophies were analyzed. Segregation of etn
spores
indicated that the psd2 allele was not lethal and resulted in
no discernable growth phenotype when expressed in the presence of the
wild type PSD1 gene. Thus, the strains from spores that were
trp
and etn
were either PSD1,
PSD2 or PSD1, psd2. Presence of the psd2 genotype was determined by backcrossing these strains with a psd1::TRP1, PSD2 strain (PTY9.2 or PTY9.4) and identifying
ethanolamine auxotrophs among sporulated progeny. The cross with the PSD1, PSD2 strain resulted in no etn
spores,
while the cross with the PSD1, psd2 strain resulted in about
25% etn
spores. This segregation of the
etn
phenotype in the resulting tetrads allowed
detection of the psd2 genotype, since only psd1::TRP1,
psd2 strains are auxotrophic for ethanolamine.
Fractionation of organelles on sorbitol gradients
was performed by a modification of the method described by Cleves et al.(18) . Cells were grown and disrupted as
described above, except the final homogenate volume was 10 ml/liter of
cells. The resulting S1.5 was then centrifuged for 15 min at 30,000
g in a Beckman JA-20 rotor. The resultant supernatant,
S30, was underlayed with 1 ml of 80% sorbitol and 1 ml of 25% sorbitol
and centrifuged for 2 h at 280,000
g
in
a Beckman SW41Ti rotor. The interface between the 25 and 80% sorbitol
layers was harvested and layered as the 50% step of a 40-80%
sorbitol gradient prepared in 10% increments of 2.5 ml each. The
gradient was developed by centrifuging in an SW41 Ti rotor at 280,000
g
for 40 h. Fractions were collected by
aspiration from the top of the gradient and stored at -20 °C.
Percoll gradient separation was carried out by a modification of the
procedure described by Cunningham and Wickner(19) . Cells (1
liter) were grown to approximately 5 10
/ml in
YPDAUE medium and spheroplasts prepared as described by Daum et
al.(15) . Spheroplasts (in 10 ml) were then layered over
10 ml of 1.8 M sorbitol, 10 mM Tris-HCl, pH 7.5, and
centrifuged for 5 min at 3000
g. The spheroplast
pellet was resuspended in 2.5 ml of 0.45 M mannitol, 10 mM Tris-HCl, pH 7.4, disrupted by 15 strokes of a tight-fitting
Dounce homogenizer, and diluted to 5 ml with additional buffer and
centrifuged 5 min at 1,200
g. The homogenization
procedure was then repeated on the pellet. The final pooled homogenate
was cleared of undisrupted cells and debris by two consecutive 1,000
g spins for 15 min each. Then 2 ml of resulting
supernatant was layered on a two-step self-forming Percoll gradient
consisting of 1 ml of 30% Percoll overlaid with 9 ml of 18% Percoll.
Both layers also contained 0.5 M mannitol, 10 mM Tris-HCl, pH 7.5. The Percoll gradients were centrifuged at 4
°C for 90 min at 40,000
g
in a
Beckman Ti50 rotor. Fractions were collected from the top of the
gradient by aspiration and stored at -20 °C.
Figure 1: Double PSD mutants require ethanolamine for growth. Viability of PSD mutant and wild type strains cultured in SD medium lacking ethanolamine (and choline) was determined by spreading culture aliquots on rich plates after the indicated times of incubation at 30 °C. Data are a representative one of four experiments.
Table 2shows the PSD activity in
mutant and wild type strains as measured by the standard and NBD-PtdSer
assay protocols. By the standard assay, cells expressing only PSD2 have <4% wild type enzyme activity, cells expressing PSD1 have 60% wild type activity, and cells with defects affecting
both loci express no detectable PSD activity. When the NBD-PtdSer
substrate is used, the relative enzyme activity of the PSD2 strain is increased 1.7-fold, activity in cells expressing PSD1 decreases to <40% of wild type, and PSD activity in
the double mutant is undetectable. Using this second method, the enzyme
activity in psd1-
1::TRP1 PSD2 cells was as high as 12% of
wild type. These data indicate that the decarboxylase activity of cells
containing wild type PSD2 and the null allele for PSD1 is significant and can account for 4-12% of the activity
found in wild type cells. It is not difficult to understand how this
second PSD activity was missed previously, since by standard methods
the activity accounts for only a very small portion of the wild type
activity. These data also demonstrate that the NBD-PtdSer substrate is
somewhat preferred by PSD2 over the standard PtdSer substrate. The
NBD-PtdSer substrate was utilized in subsequent experiments to perform
subcellular localization of the PSD2 activity (discussed later).
Figure 2:
PSD mutations affect amino phospholipid
metabolism. Incorporation of [H]serine into
phospholipids of wild type and PSD mutant strains was monitored for
cells incubated 6 h in log phase in SD plus 20 µCi/ml L-[3-
H]serine without (A) or
with (B) 2 mM ethanolamine and choline. Lipids were
extracted and separated as described under ``Experimental
Procedures.'' Values are the mean ± StdDev of six total
determinations performed in three experiments. Abbreviations are:
PtdOH, phosphatidic acid; Etn, ethanolamine; Cho,
choline.
Significant serine is incorporated into the PtdCho pool in
the double mutant despite the defect in conversion of PtdSer to PtdEtn (Fig. 2) and likely represents incorporation via the
one-carbon pool (see ``Discussion''). In addition, there is a
measurable amount of serine incorporation into PtdEtn in the double
mutant. This could be due to leakiness of the psd2 mutation.
Alternatively, serine is incorporated into sphingolipids by the action
of the enzyme serine palmitoyltransferase, and sphingolipids are broken
down to yield phosphoethanolamine, which can be utilized for PtdEtn
synthesis(29) . Fig. 3shows the effect of a
serine palmitoyltransferase inhibitor(17) , sphingofungin C
(SF-C), on the incorporation of [H]serine into
phospholipids by the PSD double mutant. In these experiments, treatment
of the cells with SF-C reduces incorporation of the labeled serine into
PtdEtn from 11% of the total to less than 3%, a level similar to that
in PtdIns. These data indicate that a significant portion of PtdEtn
label in the double mutant comes from sphingolipid breakdown rather
than a leaky psd2 mutation. In contrast to the effect of SF-C
upon [
H]serine incorporation into PtdEtn are the
effects upon PtdCho labeling. This latter result is expected if the
labeling of the PtdCho pool is derived principally from the one carbon
pool (see ``Discussion''). Thus, these data clearly
demonstrate that the ethanolamine auxotrophy of the psd1-
1::TRP1, psd2 mutant is due to a metabolic defect in
the ability to convert PtdSer to PtdEtn caused by loss of both PSD1 and
PSD2 activities.
Figure 3:
Sphingofungin C inhibits
[H]serine incorporation into
phosphatidylethanolamine by the psd1-
1::TRP1 psd2 mutant.
Cells were pretreated for 15 min with the serine palmitoyltransferase
inhibitor sphingofungin C and then incubated 6 h in L-[3-
H]serine as described under
``Experimental Procedures'' and Fig. 2. Data are the
mean ± StdDev for two separate experiments performed in
duplicate. Abbreviations are as in Fig. 2.
Figure 4:
The phosphatidylethanolamine content of psd11::TRP1 psd 2 cells is significantly reduced. Cells
were grown in log phase for greater than five generations in SD plus 2
mM ethanolamine and choline containing 10 µCi/ml
[
P]orthophosphate. Lipids were extracted and
separated as described under ``Experimental Procedures.''
Data are the mean ± StdDev from a representative two of four
experiments performed in duplicate.
The turnover of P-labeled lipids in the presence
and absence of 2 mM ethanolamine and 2 mM choline was
determined by uniformly labeling log phase cells and then shifting the
cultures to unlabeled medium (Fig. 5, A-D). In
general, inclusion of ethanolamine and choline reduced turnover of
PtdEtn but not PtdIns and reduced the accumulation of PtdCho in both
wild type and psd1-
1::TRP1, psd2 cells (compare open and filled symbols). In both media PtdSer was readily
turned over by wild type cells. In the double mutant, however, PtdSer
exhibited very low turnover and, in the presence of ethanolamine and
choline, appeared to accumulate (Fig. 5A).
PtdEtn was readily turned over in both strains (Fig. 5B) accumulating into PtdCho (Fig. 5C). Note that the lower level of PtdCho
accumulation in the double mutant is a result of the lower amount of
PtdEtn available for conversion to PtdCho (Fig. 5B). Radiolabel in PtdIns also decreased
in both strains, although the level in psd1-
1::TRP psd2 cells remained about 40% higher than that in wild type throughout
the time course (Fig. 5D). These data clearly
demonstrate the defect in conversion of PtdSer into PtdEtn in psd1-
1::TRP, psd2 cells as evidenced by both the lower
relative level of PtdEtn in these cells as well as the lack of PtdSer
turnover.
Figure 5:
Phosphatidylserine turnover is decreased
in psd11::TRP1 psd2 cells. Cells were labeled with
[
P]-orthophosphate as described in Fig. 4. They were then diluted into SD plus (solid
symbols) or minus (open symbols) 2 mM ethanolamine and choline. Samples were taken at time 0, 1, 3 and 6
h, and lipids extracted and separated as described under
``Experimental Procedures.'' Data are the mean of two
experiments performed in duplicate.
Figure 6:
PSD2 activity localizes to a different
subcellular fraction from PSD1. Cell homogenates were prepared and
separated into 30,000 g pellet and supernatants, and
PSDs or marker enzymes assayed as described under ``Experimental
Procedures.'' A, fractionation of PSD1 and PSD2 as
compared to the mitochondrial marker cytochrome c oxidase. B, comparison of PSD2 fractionation with that of various
organelle markers in psd1-
1::TRP1 PSD2 cells. Data are
the mean ± StdDev for two to five separate experiments with
duplicate determinations. Abbreviations are: PL.MEM, plasma
membrane; CCR, cytochrome c reductase; E.R.,
endoplasmic reticulum.
In an effort to determine the subcellular
location of the PSD2 enzyme, its sedimentation characteristics were
compared with markers of other organelles: plasma membrane ATPase
(plasma membrane), cytochrome c reductase (endoplasmic
reticulum), GDPase (Golgi), KEX2 protease (late Golgi), and vacuolar
ATPase (vacuoles). Fig. 6Bshows the
differential sedimentation of these markers at 30,000 g. The majority of the plasma membrane marker and a little
less than half of the endoplasmic reticulum marker sediment into the
P30 under these conditions, suggesting they are unlikely candidates for
PSD2 localization. However, it is possible that PSD2 resides in a
subpopulation of these organelles. In contrast, the Golgi and vacuolar
markers fractionate similarly to PSD2, primarily to the S30, making
these more likely possibilities.
Figure 7:
PSD2 activity codistributes with vacuolar
and Golgi membranes on sorbitol gradients. 30,000 g supernatants (S30) were prepared and separated on 40-80%
sorbitol gradients and assayed as described under ``Experimental
Procedures.'' A, distribution of PSD activity from S30s
of wild type, PSD2, and PSD1 expressing strains. B and C, distribution of marker molecules on a gradient from psd1-
1::TRP1 PSD2 cells; CCR, cytochrome c reductase, as a marker for endoplasmic reticulum; plasma membrane
ATPase as a marker for plasma membrane; KEX2, as a Golgi
marker; and vacuolar ATPase as a vacuolar marker. Data are a
representative one of three separate
gradients.
Figure 8:
PSD2 activity segregates bimodally with
vacuolar and Golgi membranes on Percoll gradients. Post-nuclear
supernatant from the psd1-1::TRP1 PSD2 strain was
separated on a self-forming Percoll gradient and markers assayed as
described under ``Experimental Procedures.'' VacATPase, vacuolar ATPase; GDPase, general Golgi
marker; KEX2, marker of late Golgi. Data are a representative
one of three gradients.
We previously cloned the PSD1 gene encoding the
mitochondrial phosphatidylserine decarboxylase enzyme of S. cerevisiae and constructed strains containing a null allele
for the gene(9) . We observed that despite the
deletion/disruption of the PSD1 gene, cells remain
prototrophic for ethanolamine, retain 2-5% of parental PSD
activity, and show only minor defects in aminophospholipid metabolism.
This led us as well as Clancey et al.(10) to
hypothesize the presence of a second PSD activity in S.
cerevisiae. The current studies demonstrate the presence of
a second, non-mitochondrial PSD enzyme activity (PSD2) in the yeast S. cerevisiae. This PSD2 enzyme activity is
sufficient to allow growth of psd1-1::TRP1 null mutants
in the absence of exogenous ethanolamine. However, mutations leading to
loss of PSD2 activity in psd1-
1::TRP1 null mutants result
in ethanolamine auxotrophy, altered aminophospholipid metabolism, and
significantly decreased levels of cellular phosphatidylethanolamine. In
addition, the PSD2 enzyme activity is not localized to the inner
mitochondrial membrane, as is PSD1, but to subcellular fractions with
properties similar to vacuoles and the Golgi apparatus.
Atkinson et al.(7) previously isolated ethanolamine auxotrophs
from mutagenized yeast. All of the mutants described carried mutations
in the cho1 gene encoding phosphatidylserine synthase.
Interestingly, none of the mutants had defects in PSD. Similar
approaches in our laboratory led to similar results. In fact, mutants
in PSD1 were only isolated when a brute force screen was used
to identify strains with low PSD activity(9) . The results
presented in the current study indicate that the likely reason for
these previous findings was the presence of a second PSD which is not
easily detected by standard assay methods. Yet, as demonstrated before (9, 10) , the activity (PSD2) is sufficient to meet
the requirement for cell growth without ethanolamine. Mutagenesis of
cells expressing only PSD2 readily results in the creation of strains
requiring ethanolamine for growth (Fig. 1), some of which
lack any measurable PSD activity (Table 2). The mutation
leading to loss of PSD2 activity segregates independently of PSD1, indicating that the genes are not linked. It is also
noteworthy that the ethanolamine requirement of strains containing both
the psd1::TRP1 and psd2 alleles is also
satisfied by choline (data not shown), supporting previous reports (6, 7, 8) that PtdCho, and not PtdEtn, is the
essential aminophospholipid end product.
Strains containing the psd1-1::TRP1 allele and lacking measurable PSD2 activity (psd2) express readily observable defects in aminophospholipid
metabolism (Fig. 2). In contrast to strains expressing
one or the other of the two PSD activities, the double mutants show
significant accumulation of [
H]serine into PtdSer
and very low levels of precursor incorporation into PtdEtn. It was
expected that since PSD activity was completely lost in the psd1-
1::TRP1, psd2 mutants that undetectable amounts of
[
H]serine would be incorporated into PtdEtn.
However, as much as 12% of the total
H recovered in lipids
was associated with PtdEtn ( Fig. 2and
3B), indicating either a leaky psd2 mutation
or an alternative pathway for radiolabeling the PtdEtn. This
alternative pathway was likely to be [
H]serine
incorporation into sphingolipids via the action of serine
palmitoyltransferase, and catabolism of the
[
H]serine labeled sphingolipids to yield
endogenous [
H]ethanolamine available for
incorporation into PtdEtn (29) . Treatment of psd1-
1::TRP1 psd2 cells with sphingofungin C, a drug
which specifically blocks serine palmitoyltransferase(17) ,
reduces incorporation of [
H]serine into PtdEtn to
essentially background levels (Fig. 3B). Thus,
the incorporation of [
H]serine into PtdEtn by the
double PSD mutant appears to be primarily a result of endogenous
ethanolamine production via sphingolipid turnover. Atkinson (31) reported that the cho1 phenotype can be
suppressed by increased production of endogenous ethanolamine most
likely via sphingolipid breakdown in eam1 mutants. Our data
indicate that the normal level of endogenous ethanolamine production is
insufficient to repress the ethanolamine auxotrophy of the PSD double
mutant. However, the eam1 mutation might be expected to
complement our psd1-
1::TRP1 psd2 mutant.
Incorporation
of [H]serine into PtdCho was comparable among the
various psd
strains. Treatment of cells with
SF-C had little effect on [
H]serine incorporation
into PtdCho by the double mutant (Fig. 3C),
suggesting that the label in PtdCho is not likely to originate from
PtdEtn labeled via the sphingolipid pathway. This is not surprising,
since Atkinson (31) reports that although significant
[
H]serine is incorporated into PtdEtn by strains
with a bypass of defects in phosphatidylserine synthase (cho1
eam1), labeling of PtdCho is much lower than in
ethanolamine-supplemented cho1 EAM1 strains. Thus, the
H observed in PtdCho is not likely to originate from
[
H]PtdEtn synthesized by endogenously produced
[
H]ethanolamine.
Atkinson et al.(7) reported significant PtdCho labeling by
[H]serine in cho1 strains supplemented
with ethanolamine and suggested that the label was likely to be
incorporated via one-carbon metabolism and methylation by S-adenosylmethionine. When [
H]serine
incorporation by the psd1-
1::TRP1 psd2 mutant was
examined in medium lacking methionine (not shown), nearly 2-fold more
radioactivity was incorporated into PtdCho as compared to wild type
cells, suggesting that the mutant may, in the absence of an active
PtdSer
PtdEtn pathway, readily shunt serine into S-adenosylmethionine biosynthesis and thus into PtdCho.
Additionally, since the cells are grown in the presence of
ethanolamine, a substantial pool of PtdEtn would be available for
methylation to PtdCho. Finally, since the
[
H]serine is labeled on the hydrogens of the
3-carbon, incorporation into PtdCho via one-carbon metabolism (i.e. the methyl groups of aminophospholipids) would be 3-fold
over-represented on a molar basis as compared to that labeled via the
common ethyl moiety of PtdSer, PtdEtn, and PtdCho.
Loss of all PSD activity has a notable effect on cellular phospholipid composition despite the presence of exogenous ethanolamine and choline. Steady-state labeling of cellular phospholipids showed that the double mutant contains only 30% of the wild type level of PtdEtn, but comparable levels of PtdCho (Fig. 4). This result occurs in the presence of 2 mM ethanolamine and choline, allowing synthesis of PtdEtn and PtdCho via the CDP base pathway. These data suggest that PtdEtn synthesized via the CDP base pathway may be more readily methylated than that produced by the PSDs. Alternatively, since choline is present and methylation may be down-regulated (see Fig. 5), it may indicate that the PSDs are important in producing a separate pool of cellular PtdEtn.
An important issue in the characterization of the PSD2 enzyme was its subcellular localization. Fig. 6clearly differentiates its localization from that of PSD1, which resides in the inner mitochondrial membrane. These data also suggest that PSD2 is unlikely to be located in peroxisomes, which have a density similar to mitochondria(32) . Fractionation of PSD2 on gradients ( Fig. 7and 8) reveals that the enzyme distribution overlaps primarily with the marker for vacuoles (>65%), but a significant proportion (<35%) co-distributes with the markers for the Golgi. At face value it may be concluded that PSD2 is localized to both the Golgi and vacuolar compartments of the cell. Alternatively, PSD2 may reside in another compartment for which there is no standardized marker, and the activity found in the more dense fractions may be this same compartment associated with some other subcellular component of higher density. Subcellular localization experiments performed in mutants in vacuolar assembly (33) may aid in discerning if PSD2 truly resides in the vacuoles.
A question which
remains unanswered is the physiological role of the PSD2 enzyme. The
different subcellular locations of PSD1 and PSD2, however, indicate
that they play disparate roles in aminophospholipid metabolism. As
described previously by us(9) , strains containing the psd1-1:TRP1 allele (i.e. expressing only PSD2
activity), exhibit a greater tendency to produce petite, rho
cells. Additional data from our
laboratory (not shown) demonstrate that cells expressing only PSD2 have
significantly less PtdEtn in the mitochondria, indicating that PtdEtn
formed by PSD2 is not efficiently transported to the mitochondria and
may account for their dysfunction. Thus, although PSD2 activity is
sufficient to meet the biosynthetic needs for cell growth, it clearly
cannot completely fulfill the requirements for organelle specific
membrane biogenesis.
These studies demonstrate the presence of a second, non-mitochondrial phosphatidylserine decarboxylase (PSD2) activity in the yeast S. cerevisiae. Loss of PSD2 activity, alone, does not result in any detectable phenotype, but loss of PSD2 as well as PSD1 results in ethanolamine auxotrophy and impaired aminophospholipid metabolism. The subcellular location of PSD2 is clearly distinguishable from the mitochondrial PSD1 and has fractionation characteristics which overlap with both vacuolar and Golgi markers. Further understanding of PSD2 awaits the isolation and characterization of the gene encoding the enzyme, examination of strains containing the null allele for the gene, and a more detailed subcellular localization utilizing immunohistochemistry. The cloning and characterization of the structural gene encoding PSD2 is described in the accompanying manuscript(34) .