(Received for publication, December 27, 1994; and in revised form, January 18, 1995)
From the
The yeast Saccharomyces cerevisiae expresses two
phosphatidylserine decarboxylase (PSD) activities which are responsible
for conversion of phosphatidylserine to phosphatidylethanolamine, and
either enzyme alone is sufficient for normal cellular growth. However,
strains containing a PSD1 null allele and a mutation leading
to loss of PSD2 activity (psd1-1::TRP1 psd2) are
auxotrophic for ethanolamine. This nutritional requirement was utilized
to isolate the gene encoding the PSD2 enzyme by complementation. The PSD2 gene encodes a protein of 1138 amino acids with a
predicted molecular mass of 130 kDa. The deduced amino acid sequence
shows significant identity (34%) to a PSD-like sequence from Clostridium pasteurianum and the yeast PSD1 (19%) at
the carboxyl end of the protein. Of particular interest is the presence
of a sequence, GGST, which may be involved in post-translational
processing and prosthetic group formation similar to other PSD enzymes.
The PSD2 amino acid sequence also shows significant homology to the
C
regions of protein kinase C and synaptotagmin. Physical
mapping experiments demonstrate that the PSD2 is located on
chromosome 7. The PSD2 gene was heterologously expressed by
infection of Sf-9 insect cells with recombinant baculovirus, resulting
in a 10-fold increase in PSD activity. The null allele of PSD2 was introduced into yeast strains by one-step gene
deletion/disruption with a HIS3 marker gene. Strains
expressing wild type PSD1 and the psd2-
1::HIS3 allele show a small decrease in overall PSD activity, but no
noticeable effect upon [
H]serine incorporation
into aminophospholipids. Strains containing both the psd1-
1::TRP1 and psd2-
1::HIS3 null alleles,
however, express no detectable PSD activity, are ethanolamine
auxotrophs and show a severe deficit in the conversion of
[
H]serine-labeled phosphatidylserine to
phosphatidylethanolamine. These data indicate that the gene isolated is
the structural gene for PSD2 and that the PSD1 and PSD2 enzymes account
for all yeast PSD activity.
Phosphatidylserine decarboxylase (PSD) ()plays a
central role in the biosynthesis of aminophospholipids by converting
phosphatidylserine (PtdSer) to phosphatidylethanolamine
(PtdEtn)(1, 2, 3, 4, 5) .
One of the salient features of the pathways for aminophospholipid
biosynthesis that include PSDs is the inherent requirement for
interorganelle transport of lipid molecules between their site of
synthesis and the locus of the decarboxylase. In eukaryotes PtdSer is
synthesized by the PtdSer synthase in the endoplasmic reticulum (ER) (6, 7) or the closely related mitochondrial-associated
membrane(8) . As described in the preceding
article(9) , yeast express two PSD enzymes that convert the
nascent PtdSer to PtdEtn, one located in the mitochondria (PSD1) (10) and the other in Golgi and vacuoles (PSD2)(9) .
Thus, the conversion of PtdSer to PtdEtn entails a transport step from
the ER or mitochondrial-associated membrane to other organelles. The
molecular mechanisms of these transport processes remain unknown.
Yeast phosphatidylserine decarboxylase 1 (PSD1) localizes to the inner mitochondrial membrane and its structural gene (PSD1) has been isolated(11, 12) . Based on the metabolic pathways and the phenotype of strains defective in PtdSer synthesis (cho1), it was hypothesized that yeast lacking PSD activity would be reliant upon the CDP-dependent biosynthesis of the aminophospholipids from exogenous ethanolamine (i.e. ethanolamine auxotrophs). Yet, when strains containing a null allele for PSD1 were constructed they exhibited no requirement for either ethanolamine or choline for growth. In addition, these strains expressed detectable PSD activity between 2-5% of wild type levels, and aminophospholipid metabolism was only affected to a minor degree(11, 12) .
The phenotype of strains
containing a null allele for PSD1 led to the proposal of a
second decarboxylase enzyme (PSD2) in Saccharomyces
cerevisiae. In the accompanying article, we describe strains
defective in PSD2 activity(9) . Strains containing the null
allele for the mitochondrial PSD1 (psd1-1::TRP1)
and a defect in the second non-mitochondrial decarboxylase activity (psd2) have undetectable PSD activity, display ethanolamine or
choline auxotrophy, and have impaired aminophospholipid metabolism. The
purpose of the studies described in this report was to: 1) isolate and
sequence the gene encoding the PSD2 enzyme, 2) map the chromosomal
location of the PSD2 gene, 3) heterologously express the PSD2 sequence, and 4) create strains containing a null allele
for PSD2 and assess the essentiality of the gene. The results
demonstrate that PSD2 encodes a newly described gene with
limited homology to PSD1. The PSD2 gene is not
essential for growth, but its corresponding null allele in the presence
of the PSD1 null allele results in strict ethanolamine (or
choline) auxotrophy and severely impaired aminophospholipid metabolism.
The plasmid YEp352 was obtained from Dr. Alex Franzusoff, University of Colorado Health Sciences Center, Denver, CO. The pCRII plasmid and the baculovirus transfer vector pVL1392 were obtained from Invitrogen. The YEp13 and YCp50 yeast genomic libraries were generously provided by Dr. W. Dowhan (University of Texas, Houston) and Dr. Vytas Bankaitis (University of Alabama, Birmingham), respectively.
The original 11-kb insert was subcloned into a 5.5-kb and two 3-kb inserts by cleavage with HindIII plus SalI and religation into the YEp352 vector. Subclones were transformed into PTY36 and only the 5.5-kb insert complemented the ethanolamine auxotrophy. When the 5.5-kb insert was subcloned into 3- and 2.5-kb inserts by cleavage with HindIII plus SphI, no complementation was observed, indicating that the SphI site is within the gene. The 5.5-kb insert in YEp352 is denoted YEp352-PSD2 throughout this article.
The presence of a disrupted chromosomal PSD2 gene was
confirmed by polymerase chain reaction analysis (PCR)(26) .
Primers flanking the deletion/disruption were constructed such that one
primer annealed to a sequence in the 5` end of the gene that was absent
from the 3.5-kb PSD2 psd2-1::HIS3 construct used for gene
disruption. This method ensures that only the chromosomal copy of the
gene is amplified, yielding a 2.4-kb fragment for the wild type gene
and a 2.6-kb fragment for the deleted/disrupted gene. Genomic DNA was
prepared from the test strains by standard methods (27) and the PSD2 locus amplified using the GeneAmp PCR kit from
Perkin Elmer Cetus for 30 cycles (94 °C for 1 min, 55 °C for 1
min, 72 °C for 1 min) in a Perkin-Elmer DNA thermal cycler.
Amplified fragments were visualized by agarose gel electrophoresis and
staining with ethidium bromide(19) .
Figure 1: The nucleotide (A) and predicted amino acid (B) sequence of the PSD2 gene. The base pair and amino acid numbering is shown to the right of the sequences. In panel A, the open reading frame begins at base pair 278 and extends to base pair 3728. A putative TATA box is indicated by the box at nucleotides 209-215, the starting ATG (base pairs 315-317) is double underlined, and the termination codon (base pairs 3729-3731) is single underlined.
Figure 2:
Comparison of the predicted PSD2 amino
acid sequence with yeast PSD1 and a PSD-like sequence from C.
pasteurianum. The amino acid sequence is aligned to maximize
homology to the 500 amino acid sequence of yeast PSD1 (11, 12) and the predicted 296 amino acid sequence of
a PSD-like gene from C. pasteurianum (CPSD). Identical amino acids are indicated by a dot. The nine amino acid stretch in PSD1 used for generation
of a degenerate oligonucleotide probe to clone the PSD1 gene
is single underlined (PSD1 amino acids 402-410). Note
the homology among the three proteins in this region. A four amino acid
sequence, LGST, in PSD1 (PSD1 amino acids 461-464), that is
likely to be involved in pyruvoyl-prosthetic group formation is double underlined. A 10 amino acid sequence in PSD2 (PSD2
amino acids 1036-1045) with identity to the C. pasteurianum protein that contains the sequence GGST which may be involved in
post-translational processing and pyruvoyl-prosthetic group formation
is also double underlined.
Despite the low overall identity of PSD2 with PSD1 and the C. pasteurianum sequence, there are a few regions of high homology that are of particular interest. First, six of the nine amino acids encoded by the degenerate oligonucleotide used for cloning PSD1, VGATNVGSI(11) , are present in the PSD2 sequence, XGAXXVGSI, at amino acids 1011 through 1019. These residues are also highly conserved in the Clostridium sequence. Second, although the putative site for autoproteolytic processing of the PSD1 protein, LGST (PSD1 amino acids 461-464), is not present in PSD2 (see PSD2 amino acids 1070-1073), there is a stretch of 10 amino acid identity of the latter protein with the C. pasteurianum protein from amino acids 1036 to 1045 which includes a similar sequence, GGST. This sequence might provide for an analogous processing event for PSD2 and the putative Clostridium PSD. Finally, there are two tyrosine-containing stretches in which all three proteins have significant identity at PSD2 amino acids 873-884 and 947-956. Overall, the sequence data and homologies support the identity of the PSD2 gene as a PSD-like enzyme.
Figure 3: The PSD2 gene is located on yeast chromosome VII. Yeast chromosomes were separated by TAFE and probed for PSD2 and the markers HXK2 and GAL4 as described under ``Experimental Procedures.'' Lane A, chromosomes hybridized with a mixture of the probes for chromosome VII (HXK2) and chromosome XVI (GAL4); lane B, hybridization with probes to PSD2 and GAL4; and lane C, hybridization with PSD2 plus HXK2.
Figure 4:
Recombinant baculoviruses harboring the PSD2 gene cause increased PSD activity in Sf-9 cells. The PSD2 gene under polyhedrin gene promoter control was expressed
in insect Sf-9 cells using the baculovirus system. The PSD activity was
measured using the NBD-[1`-C]PtdSer (black
bars) or [1`-
C]PtdSer (hatched
bars) substrates as described under ``Experimental
Procedures.'' The Sf-9 cells were either uninfected or infected
with: wild type (WT) virus, recombinant choline kinase (CKI) containing virus or recombinant PSD2 containing virus.
Data are the mean ± S.D. for five to nine separate
determinations.
Figure 5:
Physical map of the construct used to
disrupt the PSD2 locus. The 5.5-kb insert within YEp352
containing the PSD2 sequence was disrupted by the removal of a
1.5-kb BamHI fragment and its replacement with the 1.7-kb HIS3 marker gene (top). A portion of the psd2-1::HIS3 construct was then subcloned by treating
with EcoRV excising a 3.5-kb DNA fragment and TA cloning into
the pCRII vector (bottom). The numbers indicate the
size of restriction fragments in kilobases. The abbreviations for
restriction enzymes are: E1, EcoRI; E5, EcoRV; B, BamHI. The primers used for PCR
analysis are indicated by arrows at the top of the
figure.
In order to confirm the presence
of the psd2-1::HIS3 allele in the transformants, PCR
analysis was performed. PCR primers were chosen, as shown in Fig. 5(top), so that they flanked the HIS3 disruption of the PSD2 gene, and one of the primers was
unable to pair with the transforming DNA. The latter step was designed
so that PCR products would only reflect the chromosomal copy of PSD2, and the transforming DNA would not be amplified. With
these primers, amplification of the wild type PSD2 gene yields
a 2.4-kb product and the psd2-
1::HIS3 disruption yields a
2.6-kb product. Fig. 6shows agarose gel electrophoretic
analysis of the PCR products of various wild type and his
transformants. Control PCR products of plasmids containing the
complete PSD2 gene (Fig. 6, lane
a) and the psd2-
1::HIS3 construct (Fig. 6, lane b) as well as a mixture of
these two fragments (Fig. 6, lanes cand j) are shown. Fragments from PSD2 wild type
strains shown in lanes d, f, and hare 2.4 kb, as expected for the amplification from PSD2. In contrast, amplification in the his
transformants yielded fragments of 2.6 kb (Fig. 6, lanes e, g, and i), as expected for the psd2-
1::HIS3 allele. Mating, sporulation, and tetrad
analysis of these strains clearly demonstrated that the his
phenotype and the psd2
phenotype are linked
(not shown). These data demonstrate that the PSD2 allele is
disrupted in the relevant his
transformants.
Figure 6:
Disruption/Deletion of the PSD2 gene is confirmed by PCR analysis. Genomic DNA was prepared from
control and transformed strains and subjected to PCR amplification as
described under ``Experimental Procedures.'' Presence of the
wild type gene is indicated by a 2.4-kb fragment, and the
disrupted/deleted allele is indicated by a 2.6-kb fragment. Lanes: left side, molecular weight markers; a, wild type gene control; b, disrupted gene control; c, mixture of a and b; d,
untransformed psd1::TRP1 strain (PTY9.4); e,
transformed psd1::TRP1 strain (PTY42); f,
untransformed psd1-1::TRP1 strain (PTY41); g,
transformed psd1-
1::TRP1 strain (PTY44); h,
untransformed wild type strain (SEY6211); i, transformed wild
type strain (PTY43); j, mixture of a and b.
Metabolism of serine into lipid
by the strains containing the psd2-1::HIS3 was also of
particular interest, as loss of PSD would be expected to affect the
overall conversion of PtdSer to PtdEtn. Fig. 7shows the
incorporation of [
H]serine into the major
phospholipids in the absence and presence of 2 mM ethanolamine
and choline. As observed before with strains possessing a mutation
leading to loss of PSD2 activity(9) , strains expressing only
one PSD activity (PSD1 psd2-
1::HIS3 or psd1-
1::TRP1 PSD2) show little effect on incorporation of
[
H]serine into PtdSer and PtdEtn, indicating that
either PSD is sufficient for near normal aminophospholipid metabolism.
Yet when both PSD activities are absent (psd1-
1::TRP1
psd2-
1::HIS3) there is a 2-4-fold accumulation of
[
H]serine label in PtdSer and a 60-80%
decrease of label in incorporation into PtdEtn. Although strains with
the double mutation have no measurable PSD activity, they are still
capable of incorporating some [
H]serine precursor
into PtdEtn. This result is most pronounced in cells supplemented with
ethanolamine and choline but is also detectable in unsupplemented
strains. The likely source of this radiolabeling of the PtdEtn pool is
from sphingolipid catabolism (see
``Discussion'')(9) . Collectively these data indicate
that the majority of PtdEtn synthesized from a serine precursor
requires the function of both the PSD1 and PSD2 genes.
Figure 7:
Incorporation of
[H]serine into aminophospholipids is
significantly altered in strains containing both the psd1-
1::TRP1 and psd2-
1::HIS3 alleles.
Cells were grown for 6 h in log phase in minimal medium containing 20
µCi/ml L-[
H]serine in the absence (A) or presence (B) of 2 mM ethanolamine and
choline. Lipids were extracted and separated as described under
``Experimental Procedures.'' Values are the mean ±
S.D. for two experiments performed in
duplicate.
Phosphatidylserine decarboxylase plays a central role in the biosynthesis of aminophospholipids. Recent findings (9, 11, 12) have provided compelling circumstantial evidence that S. cerevisiae expresses two genes encoding PSD activity. The PSD1 gene product is mitochondrial, and the PSD2 gene product is non-mitochondrial(9) . Although PSD2 activity is low relative to PSD1, it is sufficient to support growth in the absence of ethanolamine and choline. Loss of both PSD1 and PSD2 activities, however, leads to ethanolamine auxotrophy. The current studies took advantage of the auxotrophic requirement of the double PSD mutant to isolate the structural gene encoding the PSD2 enzyme.
Several lines
of evidence support the conclusion that the PSD2 sequence is
the structural gene for the enzyme. First, the original clone was
isolated from a genomic library in the single copy, centromeric YCp50
plasmid. Thus, it is unlikely that the cloned gene is a high copy
suppressor of the psd phenotype(30) . Second,
the PSD2 clone shows significant, albeit low, homology to the PSD1 sequence (11, 12) as well as a putative
PSD sequence from C. pasteurianum
and the PSD sequence of E. coli (not
shown,(31) ). The PSD2 sequence possesses 67% identity
to a sequence of nine amino acids (VGATNVGSI) used by us to clone the
yeast PSD1 gene(11) , and this sequence is highly
conserved among the E. coli enzyme(31) , the
mammalian PSD sequence(32) , and the C.
pasteurianum
sequence, indicating that the genes are
likely to be closely related (see Fig. 2). The LGST
sequence conserved in the E. coli(31) ,
mammalian(32) , and yeast PSD1(11, 12) sequences and demonstrated to be the site of
post-translational autoproteolytic processing and pyruvoyl group
formation vital for activity of the E. coli enzyme (33, 34) is not conserved in the yeast PSD2 sequence. However, there is a region of 10 amino acid identity
between the yeast PSD2 sequence and the PSD-like sequence from C. pasteurianum
that contains the sequence GGST (Fig. 2). It is probable that this sequence represents a
site for post-translational processing of these two enzymes that is
similar to that which occurs at the LGST site found in the other PSD
proteins described above. Finally, the heterologous expression of the PSD2 clone in Sf-9 insect cells via a baculovirus vector
resulted in dramatically increased PSD enzyme activity. This activity
was inefficiently assayed using [1`-
C]PtdSer
dispersed in detergent (see Fig. 4), which is a
characteristic of PSD2 enzyme activity that we have previously
observed(9) . However, in assays employing
NBD-[1`-
C]PtdSer and no detergent, 10-fold
overexpression of enzyme activity was readily observable. The
overexpression of PSD activity directed by recombinant baculovirus
provides strong evidence to support the conclusion that the cloned PSD2 DNA sequence encodes the structural gene for this enzyme.
In previous experiments we localized the PSD2 enzyme to a subcellular compartment with fractionation properties similar to both vacuolar and Golgi compartments(9) . Therefore, it was of interest to determine whether the PSD2 sequence contained any localization signals for these two organelles in the yeast. The PSD2 sequence was examined for homology to the vacuolar targeting sequence of the repressible alkaline phosphatase found in yeast vacuolar membranes (35) , but no matches were identified. Similar homology analysis for Golgi targeting information, however, led to the identification of a stretch of amino acids(436-453) within the PSD2 sequence for which 8 of the 18 amino acids are identical to a region of the Golgi-localized KEX2 protease targeting-retention sequence(36) : the PSD2 sequence is EFDIyneDereDSdfqsK (position identities appear in capitals), as compared to the KEX2 sequence EFDIIDTDSEYDSTLDNK. The tyrosine residue (underlined) appears to be essential for Golgi localization, and although the position of the tyrosine is apparently not absolute, its presence in the context of charged residues is an important characteristic of the motif(36) . It is possible that this sequence within PSD2 may represent an organelle localization or retention signal for the Golgi.
The PSD2 amino acid sequence contains a region of about 40 residues
from 534 to 577 with high homology to the C region of a
number of protein kinase C (PKC) enzymes and the two C
regions of synaptotagmins as shown in Fig. 8. The
identities between PSD2 and these regions of PKC and synaptotagmin
range from 34 to 47%, and the homologies are between 50 and 60%. The
C
region of PKC was first identified as a region of
conserved residues among the primary structures of PKC
,
, and
(37) , and similar regions are repeated in the sequence
of synaptotagmins, integral membrane proteins of synaptic vesicles
thought to mediate Ca
-dependent
exocytosis(38) . It is well known that PKC requires the binding
of Ca
and several PtdSer molecules for
activity(39) , and Ca
and phospholipid
binding play a central role in the function of
synaptotagmins(40, 41) . The C
domains
within these proteins are considered to contain sequences at least
partially responsible for Ca
-dependent phospholipid
binding(39, 40, 41) . Although the specific
sites of PtdSer binding in PKC have not yet been identified, Reza et al.(51) have recently utilized an anti-idiotypic
monoclonal antibody to demonstrate that there is some consensus
PtdSer-binding site among the PKCs
,
, and
. Studies
examining Ca
-dependent phospholipid binding of
synaptotagmin have demonstrated that one C
domain is
sufficient(40, 41) , and deletion of a highly
conserved sequence (SDPYVKVFL) at residues 177-185 of
synaptotagmin I abolishes binding(41) . This sequence is just
upstream from the region of homology with PSD2 and is not found in
PSD2. We currently have no indication that Ca
affects
activity of PSD2 (data not shown). The C
homology domain in
PSD2 may represent a PtdSer-binding motif common to PKC and
synaptotagmin. The Ca
dependence of PtdSer binding to
PKC and synaptotagmins, however, is likely to be mediated by additional
binding domains.
Figure 8:
The PSD2 protein contains a region
homologous to the C regions of protein kinase C and
synaptotagmin. A C
-like region of the PSD2 amino acid
sequence (amino acids 534-577) is aligned to the C
regions of protein kinase C (PKC) from A. californica (PKCac)(48) , and Drosophila
melanogaster (PKCdm)(49) , rat PKC
(PKC
r) and
(PKC
r)(50) , and
repeating C
regions of rat synaptotagmins III (SYT3r), II (SYT2r), and I (SYT1r)(38) . Residue numbers are noted to
the left and right. Bold uppercase letters in the PSD2
sequence indicate residues with at least one identity among the other
sequences. Amino acid identities with PSD2 are indicated by bold
uppercase letters within each sequence. The hyphens(-)
indicate places where gaps were inserted to maximize
identity.
Another region within the PSD2 sequence shows significant homology to human retinoblastoma-associated protein 2 (RBAP-2,(42) ) and retinoblastoma-binding protein 1 (RBP-1,(43) ). This region extends from amino acid 818 to 873 of PSD2, amino acids 285-340 of RBAP-2, and amino acids 450-506 of RBP-1 (not shown). The identity with both sequences is 37%, and the homology is 51%. Both RBAP-2 and RBP-1 are proteins that were isolated based on their interaction with the retinoblastoma gene product (pRB,(42, 43, 44) ) and are likely to be involved in regulating the inhibitory effect that pRB has on cell proliferation(45) . The region of PSD2 homology does not coincide with the binding site for pRB on these proteins(42, 43) . The significance of PSD2's homology with these pRB-binding proteins is unclear, but the sequence likely serves a similar function in all three proteins.
Loss of PSD2
activity alone via disruption of the gene does not result in
ethanolamine auxotrophy, does not noticeably affect the growth of the
cells, and does not cause a defect in the incorporation of
[H]serine into aminophospholipids (Fig. 7). Disruption of both the PSD1 and PSD2 alleles, however, abolishes any measurable PSD activity (Table 2), causes the accumulation of
[
H]serine label in PtdSer and blocks most
precursor conversion to PtdEtn (Fig. 7), and results in
strict ethanolamine auxotrophy. This phenotype is indistinguishable
from that of the psd1-
1::TRP psd2 strain described by us
in the accompanying article(9) , indicating that the psd2 mutation in the original mutants is very tight. In addition, these
data also suggest that PSD1 and PSD2 constitute the total PSD activity
in yeast, since the psd1-
1::TRP1 psd2-
1::HIS3 strains are entirely dependent upon exogenous ethanolamine for
growth. The minor amount of radiolabeling of PtdEtn in the PSD double
mutant is similar to that observed in PtdSer synthase (cho1)
mutants. Previous data from Atkinson (46) as well as this
laboratory (9) suggest that PtdEtn formation in mutants blocked
in PtdSer synthesis (cho1) or decarboxylation (psd1
psd2) occurs as a consequence of sphingolipid breakdown. In the
PSD double mutants (psd1-
1::TRP1 psd2), the formation of
PtdEtn can be almost eliminated by the addition of sphingofungin C, an
inhibitor of serine incorporation into
sphingolipids(9, 47) .
Our initial goal in studying the PSD in S. cerevisiae was to begin to dissect the interorganelle transport of PtdSer from its site of synthesis in the endoplasmic reticulum to the inner mitochondrial membrane where it is converted to PtdEtn by PSD1. These studies led to the identification of the second, non-mitochondrial PSD2 (9, 11) and the isolation of its structural gene. In summary, we have isolated, sequenced, mapped, heterologously expressed, and made null alleles of the structural gene for PSD2, a newly described enzyme of aminophospholipid metabolism. The construction of null mutants in the PSD1 and PSD2 genes provides the genetic raw material for isolating mutants defective in lipid transport to the loci of the structural genes and will allow for investigation into the molecular mechanisms of these intracellular lipid transport processes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19910[GenBank].