©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphatidylserine Decarboxylase 2 of Saccharomyces cerevisiáe
CLONING AND MAPPING OF THE GENE, HETEROLOGOUS EXPRESSION, AND CREATION OF THE NULL ALLELE (*)

(Received for publication, December 27, 1994; and in revised form, January 18, 1995)

Pamela J. Trotter John Pedretti Rachel Yates Dennis R. Voelker (§)

From the Lord and Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto Clinical Research Center, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-Delta1::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(2) 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-Delta1::HIS3 allele show a small decrease in overall PSD activity, but no noticeable effect upon [^3H]serine incorporation into aminophospholipids. Strains containing both the psd1-Delta1::TRP1 and psd2-Delta1::HIS3 null alleles, however, express no detectable PSD activity, are ethanolamine auxotrophs and show a severe deficit in the conversion of [^3H]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.


INTRODUCTION

Phosphatidylserine decarboxylase (PSD) (^1)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-Delta1::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.


EXPERIMENTAL PROCEDURES

Chemicals

All simple salts, buffers, amino acids, nutritional supplements, and solvents were purchased from both Sigma and Fisher. Yeast media components (yeast extract, peptone, and nitrogen base without amino acids) were purchased from Difco. Zymolyase-100T was from ICN. The radiochemicals [3-^3H]serine, [1-^14C]serine, [S]dATP, and [P]dCTP were from Amersham Corp. and ICN. Phosphatidyl[1`-^14C]serine was synthesized from DL-[1-^14C]serine and egg CDP-diacylglycerol by the action of PtdSer synthase(13) . The 1-acyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl-Ptd-[1`-^14C]Ser (NBD-Ptd-[1`-^14C]Ser) was synthesized by the same method only using NBD-CDP-diacylglycerol prepared using the method described by Agranoff and Suomi (14) as modified by Raetz and Kennedy(15) . PtdSer synthase was purified from an Escherichia coli strain harboring the pPS3155 plasmid and overexpressing the enzyme (13) (generously supplied by Dr. William Dowhan, University of Texas, Houston, TX). Phospholipid standards for thin layer chromatography were obtained from Avanti Polar Lipids and Sigma. Thin layer Silica Gel H plates were purchased from Analtech Corp. The glass beads (0.5 mm) used for cell homogenization were purchased from BioSpec. BCA protein assay reagents were purchased from Pierce.

Yeast Strains, Plasmids, and Libraries

The wild type strain 422 (MATalpha trp1-289 lys2 his7 leu2-3, 112) was obtained from Dr. Robert Sclafani, University of Colorado Health Sciences Center, Denver, CO. The strains SEY6210 (MATalpha ura3 leu2 his3 trp1 lys2 suc2) and SEY6211 (MATa ura3 leu2 his3 trp1 ade2 suc2) were obtained from Dr. Michael Yaffe, University of California, San Diego, CA. The ethanolamine auxotrophic strain PTY36 (MATa trp1 ura3 lys2 leu2 psd1-Delta1::TRP1 psd2) was derived from a cross between the previously isolated ethanolamine auxotroph PTY18 (9) and SEY6210. PTY 9.4 (MATalpha trp1 his3 ade ura3 met14 psd1::TRP1) and PTY13 (MATalpha trp1 leu2 his lys2 psd1-Delta1::TRP1) are from our original construction of strains containing the PSD1 null allele (11) . PTY41 (MATalpha trp1 his3 ura3 lys2 leu2 psd1-Delta1::TRP1) is from a cross of PTY36 and SEY6210. The psd2-Delta1::HIS3 allele (described below) was introduced into PTY9.4, SEY6211, and PTY41 to give PTY42, PTY43, and PTY44, respectively. Yeast were cultured in synthetic or YPD media prepared by standard methods(16) . Adenine (20 mg/liter), uracil (20 mg/liter) and ethanolamine (2-5 mM) were routinely added to the YPD to give YPDAUE, and ethanolamine and choline (2 mM) supplemented media prepared as described previously(9, 11) .

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.

Assays of Phosphatidylserine Decarboxylase

Phosphatidylserine decarboxylase activity was measured using a ^14CO(2) trapping method (1) and either NBD-Ptd[1`-^14C]Ser or the standard Ptd[1`-^14C]Ser as substrates(9) . Cells were grown to log phase in YPDAUE or synthetic media as noted and harvested. Pellets were washed in water and resuspended to 0.1 g, wet weight/ml in PSD homogenate buffer containing, 0.25 M sucrose, 10 mM KPO(4), pH 6.8, 3 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol. Homogenates were prepared by agitating the cell suspension for 1 min at 4 °C with 0.5-mm glass beads in a BioSpec Mini-Beadbeater (Bartlesville, OK). Separation of the beads from the homogenate was achieved by centrifuging the homogenate through a microfuge tube punctured with a 27-gauge needle. Protein concentrations were measured using the BCA protein assay system (Pierce) in order to standardize the homogenate to 4 µg of protein/assay. To a 16 times 100-mm glass tube on ice, 10 µl of protein of the cell homogenate and 0.4 ml of the reaction mix containing 100 µl of 0.1 M KPO(4), 180 µl of PSD homogenate buffer without 2-mercaptoethanol, 120 µl of aqueous NBD-Ptd[1`-^14C]Serine (50,000-80,000 counts/min/assay at 55 µCi/µmol) were added. In some assays, denoted ``standard,'' Ptd-[1`-^14C]Ser (80,000 counts/min/assay at 0.1-0.2 µCi/µmol) dispersed in Triton X-100 (final concentration 0.1%) was used as the substrate. For assays using either substrate, the tube was capped with a stopper containing a 25-mm diameter filter paper saturated with 2 N KOH and incubated 30 min at 36 °C. The reaction was stopped by addition of 0.5 ml of 0.25 M H(2)SO(4) and incubated an additional 30 min at 36 °C. Decarboxylase activity was then determined by quantifying the amount of ^14C adsorbed to the filter using a Beckman LS-6500 liquid scintillation spectrometer. Samples were prepared in liquid scintillation fluid consisting of 0.5 ml of H(2)O and 5.0 ml of ScintiSafe Plus (Fisher).

Isolation of the PSD2 Gene

The PSD2 gene was cloned by complementation of the ethanolamine auxotrophy of the PTY36 strain described above. DNA from a yeast genomic library (YCp50-URA3) was transformed into the cells by the high efficiency lithium acetate method of Schiestl and Gietz(17) . Cells harboring plasmids were selected by growth on medium lacking uracil. Approximately 10,000 transformants were then screened for ethanolamine prototrophic clones. The complementing plasmid was recovered from the yeast and amplified in E. coli(18) . Because of the combined size of the YCp50 vector (7.9 kb) with the large size of the insert (11 kb), the plasmid was isolated from E. coli using an SDS lysis method that is recommended for plasmids of greater than 15 kb (19) . The YCp50 containing an 11-kb insert (YCp50-PSD2) was used to transform PTY36 to confirm that it resulted in acquisition of ethanolamine prototrophy. Plasmid loss experiments were performed by growing PTY36 harboring the YCp50-PSD2 (or YEp352-PSD2, described below) plasmid under non-selective conditions for greater than 30 generations, and quantitating the percentage of cells which lose both uracil and ethanolamine prototrophy.

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.

DNA Sequencing

As indicated above, the PSD2 clone has an internal SphI site, and this was used to initiate the DNA sequencing. The 3- and 2.5-kb fragments from the HindIII/SphI cleavage were subcloned into YEp352, which contains universal primer sequences (M13 and T7). Primers that anneal to these sequences were used to begin sequencing from the internal SphI site in both directions. The gene was sequenced completely in both directions using [S]dATP and the dideoxy nucleotide termination method (20) available commercially as the Sequenase system (U. S. Biochemical Corp.). The sequence was analyzed using MacVector software (IBI), and comparison of the sequence to the international data base was performed by BLAST analysis(21) .

Chromosomal Mapping

Yeast chromosome blots were prepared by transverse alternating field electrophoresis and generously provided by Dr. Kathleen Gardiner, Eleanor Roosevelt Institute for Cancer Research, Denver, CO, as described previously(11, 22) . DNA probes were labeled using [P]dCTP and the Rediprime random primer labeling kit (Amersham). An internal 1.5-kb BamHI fragment was used as a probe for PSD2. Because there was some ambiguity in assigning the chromosomal location, markers for the two candidate chromosomes were also used. The marker for yeast chromosome VII, HXK2, was excised as a 3.8-kb EcoRI fragment out of the pJJ322 plasmid (pUCHinEco+HXK2). The 1.1-kb GAL4 fragment, a marker for chromosome XVI, was cut out of the pJJ565 plasmid (pBluescript KS + GAL4). Both of these plasmids were generously provided by Dr. Judith Jaehning and Mike Woontner, University of Colorado Health Sciences Center, Denver, CO. The definitive chromosomal location of PSD2 was assigned by performing hybridizations containing PSD2 along with the marker probes. Fine structure mapping was carried out using the yeast genomic library in phage of Olson et al.(23) as described previously(11) .

Heterologous Expression of the PSD2 Gene

The PSD2 sequence was expressed in the baculovirus system as described by O'Reilly et al.(24) . A 5.5-kb EcoRI fragment containing PSD2 and 115 base pairs 5` to the translation start site was excised from the YEp352-PSD2 plasmid described above. This fragment was ligated into the EcoRI site in the polylinker of pVL1392, that is 3` of the polyhedrin promoter. A monolayer of Sf-9 cells (4.5 times 10^6 cells/75-cm^2 flask) was co-transfected with the pVL1392-PSD2 vector and BaculoGoldAutographa californica DNA (Pharmingen) using the CaCl(2) method. After 7 days of culture, the transfection supernatant containing recombinant viruses was collected. For protein expression, a fresh monolayer of Sf-9 cells (5 times 10^6 cells/25-cm^2 flask) was infected for 1 h at 27 °C (multiplicity of infection of 10) with the transfection supernatant containing PSD2 recombinant virus. The infecting medium was removed, and fresh TMNFH insect medium + 10% heat-inactivated fetal bovine serum was added. Control monolayers were either not infected, infected with wild type A. californica baculovirus, or infected with recombinant virus containing the yeast choline kinase 1 (CKI1), which will be described elsewhere. After 48 h at 27 °C, the cells were harvested, washed three times with ice-cold phosphate-buffered saline, pH 7.4, resuspended in PSD assay homogenization buffer, and disrupted by two 30-s sonication bursts on ice. The PSD activity in the preparation was assayed immediately as described above.

Construction of PSD2 Null Mutants

A 1.5-kb BamHI fragment was removed from within the PSD2 gene on the YEp352 plasmid. The HIS3 marker gene (1.7 kb) was cut out of the ZOO5 (pUC18 + HIS3) (25) plasmid with BamHI and ligated into the gap within the PSD2 sequence. A 2.5-kb blunt-ended fragment of this psd2-Delta1::HIS3 construct was excised from the YEp352 plasmid after treatment with EcoRV and cloned into a pCRII vector by TA cloning (InVitrogen). The chromosomal copy of PSD2 was then disrupted by one-step gene replacement(25) . The 3.5-kb psd2-Delta1::HIS3 construct was linearized by EcoRI digestion and used to transform strains auxotrophic for histidine (his3) using the YeastMaker system (Clonetech). Recombinants were selected by acquisition of histidine prototrophy.

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-Delta1::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) .

Phospholipid Analysis

Radiolabeled precursor incorporation into phospholipids was determined for wild type and mutant strains grown either with or without 2 mM ethanolamine and choline. Strains were grown to late log phase at 30 °C in minimal medium containing auxotrophic requirements plus ethanolamine and choline. Subsequently, the cells were washed twice in water and diluted to approximately 2 times 10^6/ml in the same medium containing 20 µCi/ml L-[^3H]serine either supplemented with or without ethanolamine and choline. Cells (1 ml) were harvested after 6 h of growth at 30 °C and combined with 10 mg of ice-cold carrier cells. The cells were isolated by centrifugation, and the pellets were washed twice with ice-cold water. The lipids were extracted and analyzed by thin layer chromatography as described previously(11) . Radioactivity associated with the different lipid classes was quantified by liquid scintillation spectrometry as described above.


RESULTS

Isolation of the Gene for Yeast PSD2

The gene encoding the yeast phosphatidylserine decarboxylase 2 was isolated by complementation of the ethanolamine auxotrophy of a psd1-Delta1::TRP1 psd2 strain(9) . After transformation with YCp50 or YEp13 yeast genomic libraries, approximately 10,000 primary transformants, each identified as uracil or leucine prototrophs, respectively, were screened for ethanolamine prototrophy. The ethanolamine prototrophs contained plasmids encoding PSD1 and PSD2, which were readily distinguished by their restriction endonuclease cleavage patterns (not shown). The PSD2 gene was first identified as complementing activity present in an 11-kb genomic DNA insert contained in the YCp50 library. The 11-kb fragment was subcloned into YEp352 using HindIII and SalI to yield a 5.5-kb insert that retained complementing activity. Growth of double mutant cells harboring either YCp50-PSD2 or YEp352-PSD2 under nonselective conditions (YPDAUE medium) for at least 30 generations resulted in coincident loss of uracil and ethanolamine prototrophy in cells (1% for YCp50 and 30% for YEp352), confirming that the PSD2 insert in the plasmids contains the complementing sequence. Table 1shows the PtdSer decarboxylase activity in wild type and mutant strains of yeast with and without complementing plasmids. As reported previously(11) , strains expressing only PSD2 (psd1-Delta1::TRP1) have approximately 5-10% of wild type PSD activity, and double mutant strains (9) with a defect in PSD2 in addition to the PSD1 null allele (psd1-Delta1::TRP1 psd2) have undetectable activity. When these double mutants harbor a single-copy centromeric plasmid, YCp50, containing the original 11-kb insert (YCp50-PSD2), their PSD activity is raised to the level of that in the PSD2-expressing strain (psd1-Delta1::TRP1). When the double PSD mutant is transformed with the multicopy YEp352 plasmid containing a 5.5-kb subclone of the original insert (YEp352-PSD2) the decarboxylase activity is about 10-fold higher than that found with the YCp50 vector. In addition, expression of the PSD2 insert on the YEp352 vector brings the PSD activity in the double PSD mutant to a level about 60% of wild type PSD activity. For the sake of comparison, the PSD activity in the double mutant transformed with a YEp352 vector containing the PSD1 gene is also included (YEp352-PSD1). Expression of PSD1 on a high copy plasmid gives PSD activity that is about 6-fold above wild type and 10-fold above the activity found in cells expressing the PSD2 clone in the same vector. The increase in PSD activity in the double mutants expressing the PSD2 insert provides evidence that the gene encodes a PtdSer decarboxylase. The clone is not likely to encode the previously isolated PSD1, since it displays a different restriction pattern (not shown) and the amount of PSD activity resulting from expression of the insert in the single-copy vector is in the range expected for the PSD2 gene (compare Table 1, second and fourth lines).



Sequence of the Yeast PSD2 Gene

The 5.5-kb insert in the YEp352 vector was utilized for sequencing of the gene. During subcloning experiments, it was observed that digestion of the 5.5-kb insert to 3- and 2.5-kb fragments with SphI yielded two subclones incapable of complementing the double PSD mutant. It was therefore concluded that the SphI site was within the gene, and DNA sequencing was performed in both directions from this site. As shown in Fig. 1A, the sequence contains an open reading frame of 3450 base pairs beginning at nucleotide 278 and ending at the in-frame termination codon at 3728. A candidate TATA box sequence is located between nucleotides 209 and 215. The likely translation start site begins at nucleotide 315. In addition, there is an A nucleotide located at position -3 from the translation start site, which is strongly preferred (75%) in yeast initiation codons(28) . The SphI cleavage site is located at nucleotide 2462. The deduced amino acid sequence (Fig. 1B) encodes a protein of 1138 amino acids with a calculated molecular mass of 130 kDa. Fig. 2shows a comparison of the deduced amino acid sequence of PSD2 to that of yeast PSD1 (11, 12) and a PSD-like sequence from Clostridium pasteurianum(^2)as revealed by a BLAST data base homology search. It should be noted that the sequence from Clostridium was identified as a PSD-like sequence due only to its homology with E. coli PSD and yeast PSD1 sequences, and not via any demonstration of encoded PSD activity. The sequences were aligned with gaps inserted in order to maximize the identity. When the entire 296 amino acid C. pasteurianum sequence is aligned with the C-terminal 352 amino acids of PSD2, 34% (101/296) of the residues are identical. The overall identity with the 500 amino acids of PSD1 is about 16% (78/500), but it reaches approximately 19% over the C-terminal 339 amino acids of PSD1 and C-terminal 288 amino acids of PSD2.


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^2 (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.

Chromosomal Mapping of the PSD2 Gene

The chromosomal location of the PSD2 gene was mapped utilizing DNA hybridization to blots of yeast chromosomes separated by transverse alternating field electrophoresis(22) . A 1.5-kb BamHI fragment of the PSD2 gene was P-labeled and used to probe the chromosomal blot, revealing a localization to either chromosome VII or XVI, which migrate very closely on the blot. In order to determine to which chromosome PSD2 actually maps, probes for marker genes to identify chromosomes VII (HXK2) and XVI (GAL4) (29) were included in the hybridization mixtures. The results are shown in Fig. 3. Lane Ashows the hybridization pattern when probes for both GAL4 and HXK2 are included in the hybridization mixture, identifying the positions of chromosomes XVI and VII, respectively. Lane Bshowing two bands when probes for both PSD2 and GAL4 are included demonstrates that these two genes are on different chromosomes. The hybridization pattern when the PSD2 and HXK2 probes are included together yields only one band (lane C), indicating that the two genes map to the same chromosome. Thus these data show that the PSD2 unambiguously maps to chromosome VII in the yeast S. cerevisiae. We also used hybridization analysis to map PSD2 at higher resolution with the phage library constructed by Olson et al.(23) . Unfortunately, the PSD2 probe failed to hybridize to any clones, indicating that the gene lies in a region of the chromosome not covered by the library.


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.



Heterologous Expression of PSD2

To verify that PSD2 is the structural gene encoding the PSD2 enzyme, we used heterologous expression of the gene in insect cells. The PSD2 sequence was placed within the genome of baculovirus under control of the polyhedrin promoter and expressed by viral infection of Spodoptera frugiperda (Sf-9) cells. PSD activity was measured using either the NBD-[1`-^14C]PtdSer or the standard [1`-^14C]PtdSer substrate and cell homogenates. Results presented in Fig. 4show the PSD activity observed in Sf-9 cells infected with the PSD2-containing baculovirus as compared to uninfected cells, those infected with wild type virus, and those infected with virus containing an unrelated gene, yeast choline kinase (CKI). Using the NBD-[1`-^14C]PtdSer substrate, which more efficiently assays the PSD2 enzyme(9) , the activity in extracts from cells infected with the PSD2 virus is elevated more than 5-fold over uninfected cells, 12-fold over wild type virus-infected cells, and 9-fold over CKI-infected cells. In the standard assay, which utilizes [1`-^14C]PtdSer dispersed in Triton X-100, activity in extracts from PSD2-expressing cells is about 2.5-fold over cells infected with wild type or CKI virus and 34% above uninfected cells. Thus, these data confirm that the PSD2 gene does encode a protein possessing phosphatidylserine decarboxylase activity and, since it raises PSD activity in this heterologous system, is very likely to be the structural gene encoding PSD2 activity.


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`-^14C]PtdSer (black bars) or [1`-^14C]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.



Disruption of the PSD2 Gene

Strains containing a null allele for PSD2 were constructed in order to determine the essentiality of the gene for growth and to provide tools for future studies of lipid transport (see ``Discussion''). The deletion/disruption construct of the PSD2 gene was prepared as described under ``Experimental Procedures'' and as outlined in Fig. 5. A 1.5-kb BamHI fragment of PSD2 was removed, and the 1.7-kb HIS3 gene was inserted into the deletion (Fig. 5, top). An EcoRV fragment containing this psd2-Delta1::HIS3 construct was then subcloned into a pCRII vector (Fig. 5, bottom). The construct present within pCRII was then linearized using EcoRI sites at either end of the insert and transformed into his3 strains possessing wild type alleles for both PSD1 and PSD2 (SEY6211), or a strain containing a psd1-Delta1::TRP1 null allele (PTY41). Cells containing the PSD2 null allele were subsequently selected by screening for his transformants.


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-Delta1::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-Delta1::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-Delta1::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-Delta1::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-Delta1::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-Delta1::TRP1 strain (PTY41); g, transformed psd1-Delta1::TRP1 strain (PTY44); h, untransformed wild type strain (SEY6211); i, transformed wild type strain (PTY43); j, mixture of a and b.



Phenotype of Strains Containing the Null Allele for PSD2

As expected from our initial experiments with strains containing a wild type allele for PSD1 and a psd2 mutation(9) , the psd2-Delta1::HIS3 allele is not lethal in the presence of a wild type PSD1 gene. However, when strains contain disruptions in both genes, psd1-Delta1::TRP1 psd2-Delta1::HIS3, they are stringently auxotrophic for ethanolamine or choline (not shown). Table 2shows the phosphatidylserine decarboxylase activity in various strains containing the psd2-Delta1::HIS3 allele. As observed previously, strains expressing only PSD2 have <10% of wild type PSD activity (compare Table 2, lines 1 and 2 to line 3). The strain carrying only the psd2-Delta1::HIS3 null allele has near wild type activity (60-80%). However, when both the psd1-Delta1::TRP1 and psd2-Delta1::HIS3 null alleles are present, no detectable PSD activity is present. These data clearly demonstrate that presence of the psd2-Delta1::HIS3 allele results in decreased PSD activity.



Metabolism of serine into lipid by the strains containing the psd2-Delta1::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 [^3H]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-Delta1::HIS3 or psd1-Delta1::TRP1 PSD2) show little effect on incorporation of [^3H]serine into PtdSer and PtdEtn, indicating that either PSD is sufficient for near normal aminophospholipid metabolism. Yet when both PSD activities are absent (psd1-Delta1::TRP1 psd2-Delta1::HIS3) there is a 2-4-fold accumulation of [^3H]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 [^3H]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 [^3H]serine into aminophospholipids is significantly altered in strains containing both the psd1-Delta1::TRP1 and psd2-Delta1::HIS3 alleles. Cells were grown for 6 h in log phase in minimal medium containing 20 µCi/ml L-[^3H]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.




DISCUSSION

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^2 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^2 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^2 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`-^14C]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`-^14C]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(2) region of a number of protein kinase C (PKC) enzymes and the two C(2) 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(2) region of PKC was first identified as a region of conserved residues among the primary structures of PKCalpha, beta, 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(2) 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 alpha, beta, and . Studies examining Ca-dependent phospholipid binding of synaptotagmin have demonstrated that one C(2) 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(2) 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(2) regions of protein kinase C and synaptotagmin. A C(2)-like region of the PSD2 amino acid sequence (amino acids 534-577) is aligned to the C(2) regions of protein kinase C (PKC) from A. californica (PKCac)(48) , and Drosophila melanogaster (PKCdm)(49) , rat PKC (PKCr) and alpha (PKCalphar)(50) , and repeating C(2) 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 [^3H]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 [^3H]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-Delta1::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-Delta1::TRP1 psd2-Delta1::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-Delta1::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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM32453 (to D. R. V.) and GM16701 (to P. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U19910[GenBank].

§
To whom correspondence should be addressed: Dept. of Medicine, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1300; Fax: 303-398-1806.

(^1)
The abbreviations used are: PSD, phosphatidylserine decarboxylase; ER, endoplasmic reticulum; NBD, 1-acyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4yl)amino]caproyl; PtdEtn, phosphatidylethanolamine; PtdSer, phosphatidylserine; kb, kilobase(s); PCR, polymerase chain reaction; PKC, protein kinase C.

(^2)
J. Meyer, unpublished results.


ACKNOWLEDGEMENTS

We thank Peggy Hammond for excellent secretarial assistance and Dr. Kathleen Gardiner for generously providing the yeast chromosome blots. We also thank Dr. Judith Jaehning and Mike Woontner for their gift of the HXK2 and GAL4 DNAs.


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