The Schizosaccharomyces pombe cho1⁺ Gene Encodes a Phospholipid Methyltransferase

Corresponding author: Susan A. Henry, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213., sh4b+{at}andrew.cmu.edu (E-mail).

Communicating editor: P. G. YOUNG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1⁺ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1⁺ gene disruption mutant (cho1) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1⁺ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

IN Saccharomyces cerevisiae, phospholipid biosynthesis and regulation has been extensively characterized (PALTAUF et al. 1992 ; CARMAN and ZEIMETZ 1996 ; GREENBERG and LOPES 1996 ). However, the pathways of phospholipid synthesis in the fission yeast Schizosaccharomyces pombe have not been analyzed extensively and little is known about their regulation. An initial characterization of the pathways for the synthesis of phospholipids in S. pombe was reported by FERNANDEZ et al. 1986 (Figure 1). The membranes of S. pombe contain a typical mixture of phospholipids similar to that of S. cerevisiae (PALTAUF et al. 1992 ; GREENBERG and LOPES 1996 ) and other eukaryotes (KENT 1995 ).

View larger version (12K): In this window In a new window Download PPT slide   Figure 1. The phospholipid biosynthetic pathway in S. pombe. The pathway for phospholipid biosynthesis in S. pombe was described by FERNANDEZ et al. 1986 and is similar to pathways used by other eukaryotes. However, S. pombe laboratory strains are auxotrophic for inositol and do not express inositol 1-phosphate, which catalyses the first committed step to phosphatidylinositol (PI) synthesis. The S. pombe genes involved in the synthesis of PC via the PE methylation pathway are shown in boldface and underlined. The analogous S. cerevisiae genes are shown in parentheses in italics.

Unlike S. cerevisiae, however, the commonly used laboratory strains of this organism require inositol for growth and cannot synthesize inositol 1-phosphate from glucose 6-phosphate (FERNANDEZ et al. 1986 ; MAJUMDER et al. 1997 ). In S. cerevisiae, the INO1 gene, which encodes inositol 1-phosphate synthase, is highly regulated. Many mutants of S. cerevisiae defective in synthesis and/or regulation of phospholipid biosynthesis were first identified by screening for inositol auxotrophs (Ino^-) or mutants that overproduce inositol (Opi^-) (SWEDE et al. 1992 ; HENRY and PATTON-VOGT 1998 ). Because of the lack of inositol 1-phosphate synthase activity in S. pombe, neither of these screens is possible. We, therefore, elected to begin the genetic exploration of the pathways for the synthesis of phospholipids in S. pombe by screening for choline auxotrophs.

Choline auxotrophs have been isolated in a number of fungal microorganisms, including Neurospora crassa (HOROWITZ 1945, HOROWITZ 1946 ). The chol-1 mutants of N. crassa are defective in the first methylation step from phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PMME), while the chol-2 mutants are defective in the last two methylation steps: PMME to phosphatidyldimethylethanolamine (PDME) to phosphatidylcholine (PC) (HUBBARD and BRODY 1975 ). In S. cerevisiae, several independent screens for choline auxotrophic mutants resulted only in the identification of mutants that will grow in the presence of either ethanolamine or choline. These mutants represent a single complementation group, cho1, and proved to be deficient in phosphatidylserine biosynthesis (ATKINSON et al. 1980A , ATKINSON et al. 1980B ; KOVAC et al. 1980 ; HENRY 1982 ). S. cerevisiae mutants defective in the phospholipid-specific N-methyltransferases have been isolated (see Figure 1 for the gene enzyme relationships), but are not choline auxotrophs, although they grow more rapidly if supplied with choline (GREENBERG et al. 1983 ; SUMMERS et al. 1988 ; KODAKI and YAMASHITA 1989 ; MCGRAW and HENRY 1989 ; HENRY and PATTON-VOGT 1998 ).

We now report the isolation and characterization of choline auxotrophic mutants of S. pombe that are defective in phospholipid biosynthesis via the PE methylation pathway. These mutants fall into two complementation groups, cho1 and cho2. We also report the cloning and the characterization of the cho1⁺ gene, the first gene encoding a phospholipid biosynthetic enzyme to be isolated from the fission yeast.

	MATERIALS AND METHODS

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Strains, media, and growth conditions:
The strains of S. pombe used in this study and their sources are described in Table 1. Choline auxotrophs were isolated in 972, the original h^-S strain (MUNZ et al. 1989 ), or in a derivative of that strain, SH621.

Strains were maintained on the standard complex medium (YE) recommended by GUTZ et al. 1974 , which contained 0.5% Bacto-yeast extract (Difco, Detroit, MI) and 3% glucose. The medium (YEA) was further supplemented with choline (1 mM) and adenine (75 mg/ml) to support growth of strains containing cho1 and cho2 mutations and ade6 or ade7 mutations, respectively. Minimal medium (SD) consisted of 0.67% Bacto-yeast nitrogen base without amino acids (Difco), 2% glucose, 1 ml/liter of a vitamin mix (10 g/liter nicotinic acid, 1 g/liter calcium pantothenate, and 5 ml/liter of a 2 mg/ml biotin solution made in 50% ethanol), 4 ml/liter of 10 mM solution of myo-inositol, and 250 mg/liter of the following five supplements: lysine, choline, adenine, uracil, and histidine. For plates, 20 mg/liter of Magdala red (MR) (phloxin B; Sigma, St. Louis, MO) was added to facilitate the scoring of nutritional markers, particularly the choline auxotrophic mutations. This dye stains dead and dying cells magenta (GUTZ et al. 1974 ). For liquid media, 0.5 g/liter asparagine was added to prevent lysis during the final cell division before stationary phase (JOHNSON 1967 ).

The Escherichia coli DH5 cells were cultured in Luria broth medium containing 50 µg/ml ampicillin for the selection of plasmids.

Isolation and genetic characterization of choline auxotrophs:
Standard genetic techniques for S. pombe (GUTZ et al. 1974 ; KOHLI et al. 1977 ; GYGAX and THURIAUX 1984 ) were used for strain constructions, mating type determinations, genetic mapping, and other genetic procedures. The S. pombe strain SH621 (Table 1) was mutagenized with ethyl methanesulfonate, and survivors were plated for single colonies on YE and then replicated to defined medium lacking choline (SD-C)containing MR. Three colonies that grew on SD-C were identified, but they were dark red because of a large number of dead or dying cells that stained with MR. The presence of MR in the plates was crucial for visualizing choline auxotrophy. The choline-auxotrophic strains were classified by pseudocomplementation tests (GUTZ et al. 1974 ). Linkage was determined using the formulas of PERKINS 1949 .

Plasmids:
A library of S. pombe genomic DNA was generously provided by Paul Young. pCHO-1 is the original 6.4-kb HindIII cho1⁺ plasmid from that library, which was constructed in the plasmid pWH5. Plasmid pWH5 was constructed by WRIGHT et al. 1986 . pMK501 contains the 6.4-kb HindIII fragment cloned into plasmid pRS305 (SIKORSKI and HIETER 1989 ). pMK301 contains a 3.8-kb PstI-SalI fragment from pMK501 in the plasmid pIRT2. pMK302 contains a 1.4-kb PstI fragment from pMK501 cloned into plasmid pIRT2. pMK303 contains a 1.2-kb PstI fragment from pMK501 (containing the entire cho1⁺ open reading frame) cloned into plasmid pIRT2. pMKR1 is a NdeI-SmaI fragment containing the rat liver PE methyltransferase (PEMT) cDNA cloned into plasmid pREP1 (MAUNDRELL 1993 ). Plasmid pMKR1 was constructed as follows: restriction sites were placed at the 5' and 3' ends of the rat liver cDNA by PCR. Primer MKS3 (5'-CAT ATG AGC TGG CTG CTG GGT TAC GT-3') introduced a NdeI site before the initiation codon and primer MKS4 (5'-CCC GGG TCA GCT TTT GTG CAA CC-3') introduced a SmaI site at the termination codon. pMKC1 is a 1.2-kb NdeI-BamHI fragment containing the cho1⁺ open reading frame cloned into plasmid pREP1. Plasmid pMKC1 was constructed as follows: restriction sites were placed at the 5' and 3' ends of the cho1⁺ open reading frame by PCR. Primer MKS1 (5'-GAC GTC GTT CAT ATG AGC TTG ATT C-3') introduced an NdeI site before the initiation codon, and primer MKS2 (5'-TAG CTT GGA TCC TAT CAC GAG CAG CG-3') introduced a BamHI site at the termination codon. The pREP1 plasmid is an S. pombe expression vector containing the repressible nmt promoter (MAUNDRELL 1993 ). pMK68 is the 1.2-kb PstI cho1⁺ fragment cloned into pGEM2. The disruption plasmid pMK68-2 was made as follows: A HindIII restriction site was added to plasmid pMK68 that simultaneously removed 79 bp of the coding region using inverse PCR. Primer MKDH3 (5'-TAC AAG CTT CTA GCA TGT AAC AGG C-3') and primer MKDH4 (5'-TGA AGC TTC TTG TGC AGG GTA TAG C-3') introduced HindIII sites in the cho1⁺-coding region. The ura4⁺ gene was isolated by digestion of plasmid pJK210 with HindIII and inserted into the HindIII site of pMK68-2. The disruption fragment was linearized by digesting with PstI and PvuII from plasmid pMK68-2.

DNA sequence determination and analysis:
DNA sequencing of the S. pombe cho1⁺ gene was performed using the dideoxy nucleotide sequencing method (SANGER et al. 1977 ). The 1.2-kb PstI genomic fragment (pMK68) was cloned into pGEM2 (Promega, Madison, WI) to be sequenced. The primers used in this study were the universal primers (SP6 or T7) or synthetic oligonucleotides homologous to the 1.2-kb genomic PstI fragment. The DNA sequence was determined at least once for both strands. Sequencing reactions were fractionated on 6% polyacrylamide at a constant 90 W. Sequencing gels were dried directly on filter paper (3M; Whatman, Clifton, NJ) and exposed to X-ray film for 1–2 days at room temperature. DNA and protein sequence analyses were carried out using the DNA Strider computer programs. Database searches were carried out using the BLAST network service at the National Center for Biotechnology Information (ALTSCHUL et al. 1990 ). Analysis of the hydrophobicity for the cho1⁺ gene product was conducted using the algorithm of KYTE and DOOLITTLE 1982 , as executed in the DNA Strider program.

Nucleotide sequence accession number:
The DNA sequence of the cho1⁺ gene has been submitted to GenBank databases (accession no. AF004112).

Construction of an S. pombe cho1 disruption allele:
Cells of a haploid wild-type strain FY72 were transformed with the cho1::ura4⁺ DNA fragment and were spread on plates without uracil. Three independent transformants were selected and found to have a Cho^- phenotype. Southern blot analysis showed that the cho1⁺ gene was properly disrupted (data not shown). To confirm that disruption of the cho1⁺ gene has no lethal effect and that choline auxotrophy and the disruption are tightly linked, the cho1 strain was crossed with a ura4 strain and tetrad analysis was performed.

Phospholipid labeling:
Pulse labeling of cells with [methyl-¹⁴C]methionine was carried out using the procedure used for S. cerevisiae (LOEWY and HENRY 1984 ) with minor modifications. Cultures were grown overnight in synthetic defined medium plus choline (SD+C) to late log phase or early stationary phase and counted for viable cells after diluting with MR to a final concentration of 2 mg/ml. Cells were washed once with 5 ml of SD containing the necessary nutritional supplements plus various supplements: 1 mM ethanolamine, adjusted to pH 7 with HCl, 1 mM monomethylethanolamine (MME), 1 mM dimethylethanolamine (DME), and 1 mM choline (C), as described in the individual experiments. The cells were then resuspended in 5 ml of the same medium to give a final concentration of 5 x 10⁵ to 2 x 10⁶ per ml and divided into two cultures. One of the cultures was placed in a tube to follow the growth kinetics of the strain; in preparation for labeling, the other culture (5 ml) was placed in a 13 x 100-mm, screw-capped, disposable test tube. After 6–9 hr of growth (two to three generations in SD at 30°), 5 mCi/ml [methyl-¹⁴C]methionine was added to each culture to be labeled, and the cultures were incubated for an additional 30 min at 30°. An aliquot from the corresponding unlabeled culture was taken to determine viable cells. After 30 min, the cells in the labeled culture were pelleted in a clinical centrifuge at 3000 rpm.

For analysis of the phospholipid composition of various strains under a variety of growth conditions, cells were grown and prepared for labeling as described above, but were labeled to steady state, approximately six generations, in the presence of 50 µCi/ml [³²P]orthophosphate (New England Nuclear, Boston, MA). To determine terminal steady-state phospholipid composition of Cho^- strains grown in the absence of choline, cells were first grown and labeled with ³²P, as described above, in the presence of choline for at least six generations. They were harvested in midlogarithmic phase, washed twice in medium lacking choline, and then resuspended in medium lacking choline and containing ³²P at the same specific activity.

Phospholipid extraction and chromatography:
The phospholipids were extracted by methods previously described for S. cerevisiae by ATKINSON et al. 1980A and subsequently used for S. pombe (FERNANDEZ et al. 1986 ). Phospholipids were separated on SG81 chromatography paper that had been cut into 5.5 x 6.6-in pieces and dipped in 2% EDTA, pH 7.6. After the papers had air dried, they were baked at 45° for 15–20 min. For the ¹⁴C-labeled methylated phospholipids, the one-dimensional system of WAECHTER and LESTER 1971 was used. For the ³²P-labeled phospholipids, the two-dimensional system of STEINER and LESTER 1972 was used.

The air-dried chromatograms were placed on Kodak X-OMAT AR film and exposed at room temperature. The autoradiograms were used to identify the positions of the individual phospholipids, which were then cut out and counted in a liquid scintillation counter.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and characterization of choline auxotrophic mutants:
Three choline auxotrophic mutants (SF1, SF2, and SF3) were isolated in our laboratory by Serafin Fernandez (see dedicatory footnote) using the mutagenesis and screening procedures described in MATERIALS AND METHODS. In addition, Professor J. Kohli provided us with six Cho^- strains (JK2 and JK4–JK8) that had been isolated a number of years ago by Professor U. LEUPOLD (unpublished data). All nine mutants were crossed pairwise and subjected to pseudocomplementation and free spore analysis. This analysis indicated that the mutants belonged to two unlinked complementation groups. The first complementation group, cho1, consisted of two strains (JK6 and SF3), while the second, cho2, consisted of the remaining seven strains (SF1, SF2, JK2, JK4, JK5, JK7, and JK8).

The choline auxotrophic mutants were further characterized with respect to their ability to grow on minimal plates containing the phospholipid precursors ethanolamine, MME, DME, or choline. All the mutants in both complementation groups proved to be stringent choline auxotrophs and stopped growing entirely within two to three doubling times after the shift to medium lacking choline. None of the mutants could grow when supplemented with ethanolamine, suggesting that they are not analogous to the S. cerevisiae cho1 mutants. The S. pombe cho1 strains could grow only when supplemented with DME or choline. The cho2 strains could grow when MME, DME, or choline was present in the medium.

The remainder of this report will focus on the isolation and characterization of the cho1⁺ gene and characterization of the biochemical defect in the cho1 strains.

Genetic mapping of the cho1⁺ gene:
Free spore analysis indicated that the cho1⁺ gene was linked to both ade7 and ura5 on the left arm of chromosome II (data not shown). The chromosomal map position of the S. pombe cho1⁺ gene on chromosome II was then confirmed by tetrad analysis. A diploid strain was generated by mating S. pombe strains SH622 (h⁺cho1-6) and SH623 (h^-ade7-50, ura5-294). This strain was sporulated, and tetrad analysis was performed to determine the relative order and map distances between cho1-6, ade7, and ura5 (Table 2). These data indicated that relative order of these genes on chromosome II was as follows: centromere-ura5-ade7-cho1. The distance between the ura5 and ade7 loci was approximated to be 23.7 recombination units, while the distance between the ade7 and cho1 loci was ~39.5 recombination units.

Cloning of the S. pombe cho1⁺ gene:
Strain SH617, containing the S. pombe cho1 mutation, was transformed with an S. pombe genomic library, as described in the MATERIALS AND METHODS, selecting for Leu⁺ transformants. Approximately 15,000 individual leucine prototrophs were screened for their ability to grow on choline-free plates. Two potential cho⁺ plasmids were obtained and found to have overlapping inserts. Restriction analysis indicated that pCHO-1 contained a 6.4-kb insert. Retransformation of strain SH617 with pCHO-1 again rendered it cho⁺. Further subcloning was done to define the smallest complementing DNA fragment. A 3.8-kb PstI-SalI fragment, a 1.4-kb PstI fragment, and a 1.2-kb PstI fragment were subcloned into an S. pombe vector, pIRT2, generating plasmids pMK301, pMK302, and pMK303, respectively (Figure 2). After transformation into S. pombe, only plasmid pMK303 was able to complement a cho1 mutation.

View larger version (8K): In this window In a new window Download PPT slide   Figure 2. Subcloning of the S. pombe cho1+ gene. The S. pombe cho1+ gene was isolated as a 6.4-kb fragment of genomic DNA. Unique restriction sites are shown above the horizontal line. The box denotes the open reading frame, and the arrow denotes apparent start of translation. +, ability to complement the cho1 mutant strain; -, inability to complement.

Sequence analysis of the S. pombe cho1⁺ gene:
The nucleotide sequence of both strands of the 1200-bp complementing fragment on plasmid pMK68 was determined using the Sanger dideoxy method. Computer-assisted inspection of the sequence using the DNA Strider program revealed one continuous open reading frame with no apparent introns, potentially encoding a 204-amino-acid protein, starting with an ATG codon at nucleotide 429 and terminating with a TAA codon at nucleotide 1043 (Figure 3A).

View larger version (34K): In this window In a new window Download PPT slide   Figure 3. Sequence of the S. pombe cho1+ gene. (A) Nucleotide sequence and the deduced amino acid sequence of cho1+. Numbering of the amino acid residues starts with the first ATG codon, ends with the first stop codon TAA, and is highlighted in boldface. Potential TATA boxes are indicated in bold and underlined. The GenBank accession no. for this sequence is AF004112. (B) Hydropathy analysis of the S. pombe cho1+ gene product using the method of KYTE and DOOLITTLE 1982 .

The DNA sequence of the cho1⁺ gene was subjected to computer-assisted analysis as described in MATERIALS AND METHODS. Hydropathy analysis of the deduced amino acid sequence using the method of KYTE and DOOLITTLE 1982 revealed several hydrophobic stretches, suggesting that the putative S. pombe cho1⁺ gene product is an integral membrane protein (Figure 3B). The predicted size of the protein encoded by this open reading frame was 22,800 D. The cho1⁺ gene was found to hybridize with a single RNA band of ~1 kb (data not shown). This size is in agreement with the translation product of the cho1⁺ gene.

Through the use of the BLAST network service at the National Center for Biotechnology Information (ALTSCHUL et al. 1990 ; Figure 4), the S. pombe cho1⁺ gene product was found to have significant sequence homology with proteins encoded by the rat and S. cerevisiae phospholipid methyltransferases. Overall, the protein encoded by the cho1⁺ gene is 51 and 41% identical to the S. cerevisiae and rat phospholipid methyltransferases, respectively. Striking homology of the three phospholipid methyltransferases was detected in the region spanning amino acid residues 120–170 of the S. pombe cho1⁺ product. The alignment revealed ~67% homology among the three methyltransferases in this region.

View larger version (51K): In this window In a new window Download PPT slide   Figure 4. Multiple alignment of the S. pombe cho1+ gene product (cho1p) to other phospholipid methyltransferases. The amino acids that are conserved among the phospholipid methyltransferases are indicated by the black boxes. The region that is underlined is believed to be the phospholipid binding site. The proposed binding site for SAM is shown by the gray box.

Disruption of the S. pombe cho1⁺ gene:
The S. pombe cho1⁺ gene was disrupted using the one-step gene disruption technique (ROTHSTEIN 1983 ), as described in MATERIALS AND METHODS. The S. pombe ura4⁺ cassette replaced 79 bp of the S. pombe cho1⁺ coding sequence, and this linearized fragment was used to replace the cho1⁺ gene in haploid yeast strain FY72 to create the disruption mutation. The cho1 disruptant strain was viable and displayed the Cho^- phenotype. When cho1 was crossed to the cho1-6 mutant, the Cho^- phenotype segregated 0:4 in all nine tetrads, indicating linkage of the inserted DNA to cho1-6, but not to ura4. When the cho1 strain was crossed to a ura4, Cho⁺ strain, the Ura⁺ and Cho⁺ phenotypes cosegregated, confirming that the cloned DNA was the cho1⁺ gene. Southern blot analysis confirmed that the disruption had occurred at the cho1⁺ locus (data not shown). The cho1 strain was phenotypically and biochemically indistinguishable from the original cho1 mutant strains.

Phospholipid synthesis in the cho1 mutant strains:
S. pombe cho1 and cho1⁺ cells were labeled to steady state using [³²P]orthophosphate as described in MATERIALS AND METHODS to determine the total membrane phospholipid composition. The relative percentages of the phospholipids of wild-type and cho1 strains are shown in Table 3. In wild-type cells, PC represented ~40% of total phospholipid when cells were grown in the presence or absence of choline or DME. However, in cho1 cells, the relative percentage of PC was reduced when they were grown in the presence of DME. Under these growth conditions, the levels of PDME approached 47% of total phospholipid, suggesting that this lipid can substitute for PC in the membrane and support growth. However, the mutant is apparently unable to convert PDME to PC.

The terminal phospholipid composition of cho1 and cho1-6 cells was also determined after a shift to choline free medium, as described in MATERIALS AND METHODS. Under these circumstances, the cho1-6 and cho1 cells accumulated 16 and 13% PMME, respectively, before growth ceased.

Lipids were also pulse labeled with [methyl-¹⁴C]methionine (Table 4). Because the methylation reactions leading from PE to PC use S-adenosyl-methionine (SAM) as a methyl donor, [methyl-¹⁴C]methionine specifically labels the phospholipids PMME, PDME, and PC. Labeling of neutral lipids as shown in Table 4 most likely results from the methylation of sterols (LOEWY and HENRY 1984 ). Approximately 60% of the lipid-associated methyl-¹⁴C label was incorporated into PC when the S. pombe wild-type cells were grown in the absence of choline. However, in the presence of choline, only ~5% of the lipid-associated label was incorporated into PC (Table 4) and the rest accumulated in the neutral lipid fraction. Very little label was incorporated into PC in the S. pombe cho1 and cho1-6 mutant cells that were grown in the presence of choline or DME (Table 4).

Functional conservation of the phospholipid methyltransferases:
The rat liver PEMT cDNA, generously provided by Dr. Dennis Vance (CUI et al. 1993 ), was cloned into a LEU2-bearing plasmid (pREP1) that contained the S. pombe nmt1 promoter. The nmt1 gene is involved in the thiamine biosynthetic pathway and is highly regulated by the availability of thiamine (MAUNDRELL 1993 ). Expression of the rat liver PEMT gene under the control of the S. pombe nmt1 promoter alleviated the choline auxotrophy in an S. pombe cho1 mutant strain (Figure 5). In addition, PC biosynthesis was restored when the rat clone was transformed into an S. pombe cho1 mutant strain, as shown by the in vivo pulse labeling with [methyl-¹⁴C]labeled methionine (Table 4). The S. cerevisiae OPI3 gene was also cloned into the S. pombe LEU2 plasmid pIRT2. After transformation of the S. cerevisiae OPI3 gene into the S. pombe cho1 mutant strains, choline prototrophy was restored (data not shown).

View larger version (79K): In this window In a new window Download PPT slide   Figure 5. Complementation of the rat liver PEMT cDNA when transformed in an S. pombe cho1 mutation. The S. pombe cho1 mutant cells were transformed with the rat liver PEMT cDNA when placed behind the control of the S. pombe nmt promoter. Figure 5 shows an S. pombe wild-type strain, cho1 strain, and a cho1 strain expressing the rat liver cDNA (top to bottom). Leu+ transformants were patched onto selective plates, incubated at 30° for 3 days, and then replica plated to choline-free plates.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We report the isolation and characterization of choline auxotrophic mutants of the fission yeast S. pombe and the isolation of cho1⁺, the first gene encoding a phospholipid biosynthetic enzyme to be isolated in this organism. The availability of the Cho^- mutants and the isolation of the cho1⁺ gene prepares the way for genetic and regulatory studies of phospholipid biosynthesis in S. pombe comparable to those that have been carried out in the budding yeast S. cerevisiae (HENRY and PATTON-VOGT 1998 ). All eukaryotic organisms characterized to date appear to have two routes to PC biosynthesis (KINNEY 1993 ; KENT 1995 ). In mammals, the predominant route involves the reaction of cytidine diphosphate (CDP)-choline with diacylglycerol via the CDP-choline pathway (Figure 1; KENNEDY and WEISS 1956 ). In S. cerevisiae, this pathway is used for incorporation of exogenous choline into PC as well as salvage of choline liberated by PC turnover (PATTON-VOGT et al. 1997 ). A second route to PC biosynthesis involves the three-step methylation of PE to form PC, a pathway that was originally discovered in the liver by BREMER and GREENBERG 1961 .

The data presented in this report indicate that the S. pombe cho1 and cho2 mutants are defective in PC biosynthesis via the PE methylation pathway and have biochemical defects analogous to those in the S. cerevisiae opi3 (pem2) and cho2 (pem1) mutants, respectively (KODAKI and YAMASHITA 1987 ; SUMMERS et al. 1988 ; MCGRAW and HENRY 1989 ; KANIPES and HENRY 1997 ). The S. pombe cho1 mutants, like the S. cerevisiae opi3 mutants (MCGRAW and HENRY 1989 ), accumulate PMME when starved for choline or DME. When supplied with DME, S. pombe cho1^- mutants produce PDME and cannot methylate it to form PC. Furthermore, they cannot use MME to support growth. These data indicate that the final two methylation steps are defective in cho1 mutants, as is the case in the S. cerevisiae opi3 (pem2) mutants (KODAKI and YAMASHITA 1987 ; MCGRAW and HENRY 1989 ). The S. pombe cho2 mutants can use MME, DME, and choline as growth supplements, suggesting that they are defective only in the first methylation, similar to the S. cerevisiae cho2 (pem1) mutants (KODAKI and YAMASHITA 1987 ; SUMMERS et al. 1988 ).

Similar growth defects were detected in N. crassa mutants, which also fell into two complementation groups (cho1-1 and cho1-2; CROCKEN and NYC 1964 ; SCARBOROUGH and NYC 1967 ; HUBBARD and BRODY 1975 ) with defects analogous to the S. cerevisiae cho2 (pem1) and opi3 (pem2) mutants, respectively. In Aspergillus nidulans, three classes of choline auxotrophs have been reported (MARKHAM and BAINBRIDGE 1979 ). Mutants for two of the A. nidulans complementation groups, choA and choC, have growth requirements analogous to the S. pombe cho2 and cho1 mutants, respectively. Mutants representing the third complementation group, choB, resemble the S. cerevisiae cho1 mutants in that they will grow in the presence of ethanolamine, as well as MME, DME, or choline. These comparative genetic data suggest that fungi, as a group, possess two structural genes encoding phospholipid N-methyltransferases, one of which catalyzes the first methylation from PE to PMME, while the other is a bifunctional enzyme catalyzing the two-step reaction series PMME PDME PC. Mammals (RIDGWAY and VANCE 1987 ; CUI et al. 1993 ) and plants (KINNEY 1993 ), in contrast, appear to have only a single phospholipid methyltransferase activity that can carry out all three methylations, including the first step, PE PMME.

The S. pombe cho1 and cho2 mutants are choline auxotrophs, unlike the analogous S. cerevisiae opi3 and cho2 mutants (SUMMERS et al. 1988 ; KODAKI and YAMASHITA 1989 ; MCGRAW and HENRY 1989 ; GRIAC et al. 1996 ; HENRY and PATTON-VOGT 1998 ). When S. pombe cho1 cells are shifted to choline-free medium, growth stops within about two generations, at which point the membrane phospholipid composition contains ~13% PMME, 1.3% PDME, and 30% PC (Table 3). In contrast, the S. cerevisiae opi3 mutants continue growing with 44–50% PMME, 2–5% PDME, and no detectable PC (MCGRAW and HENRY 1989 ). This comparison indicates that S. pombe cells are far less tolerant of substitutions among the methylated phospholipids than are S. cerevisiae cells.

The data presented in Figure 4 suggest that the structural features of the phospholipid methyltransferases are conserved across two remotely related yeast species, S. pombe and S. cerevisiae, which are believed to have diverged perhaps 1000 mya (SIPICZKI 1989 ). The conserved features of phospholipid methyltransferase must have evolved before the divergence of the lineages leading to the fungi and the metazoans because the rat cDNA gene is also quite similar in function to the two yeast genes (Table 4). The S. pombe cho1⁺ gene product is ~51 and 41% similar to the S. cerevisiae and rat/mouse phospholipid methyltransferases, respectively. This sequence similarity is observed throughout the entire length of the 205-amino-acid protein. However, a region of high sequence identity is concentrated to a stretch of 48 amino acids between the middle and end of the protein from S. pombe amino acids 120–170, suggesting a possible conserved functional domain. Overall, relative to the other methyltransferases, there are ~66% conserved amino acids in this region, which has previously been identified as a possible binding site for the phospholipids PMME and PDME (KODAKI and YAMASHITA 1987 ; CUI et al. 1995 ).

It has been speculated that all methyltransferases, including those that catalyze methylations of DNA, protein, RNA, and lipid, contain a tripeptide SAM-binding site, GXG (INGROSSO et al. 1989 ). A very similar motif was identified in an analysis of the alignment of the S. cerevisiae and mammalian phospholipid methyltransferases (KODAKI and YAMASHITA 1987 ; CUI et al. 1995 ). However, this tripeptide motif is not conserved in the S. pombe cho1⁺ gene. In the region where the GXG motif is found in the other phospholipid methyltransferases, the multiple alignment of the S. pombe cho1⁺ gene product with other phospholipid methyltransferases identified a CFG tripeptide. This site could be the SAM-binding site for the S. pombe enzyme, but, if so, it is quite divergent from the sites reported in the other genes. The functions of these putative domains and motifs, however, have not been tested biochemically for any phospholipid methyltransferase.

The rat liver phospholipid methyltransferase can complement the S. pombe cho1 mutation, suggesting that the function and structure of phospholipid methyltransferases have been conserved between rat and yeast. S. pombe can, thus, serve as a host for structure-function studies of the mammalian phospholipid methyltransferase. This is of particular interest because the mammalian phospholipid methyltransferase may have tumor-suppressing activity (TESSITORE et al. 1997 ; VANCE et al. 1997 ). The analogous S. cerevisiae opi3 mutant is not a convenient host for assaying the function of heterologous phospholipid methyltransferases because opi3 mutants are not choline auxotrophs.

Our studies suggest that S. pombe is an excellent organism in which to study phospholipid biosynthesis, and, at the present time, it is the only eukaryotic microorganism available for convenient structure-function studies of the mammalian phospholipid N-methyltransferase. In addition, S. pombe provides a eukaryotic system second only to S. cerevisiae in the powerful genetic and molecular tools that can be applied to studies of regulatory phenomena. Preliminary results concerning regulation of phospholipid biosynthesis in S. pombe suggest that future studies will be fruitful. In wild-type S. pombe cells, incorporation of [methyl-¹⁴C]methionine into PC was dramatically reduced when the cells were grown in the presence of choline, indicating that there may be a high degree of regulation occurring in the pathways for phospholipid biosynthesis in S. pombe (Table 4). Much of the regulation in response to phospholipid precursors in S. cerevisiae occurs at the transcriptional level in response to inositol and choline (PALTAUF et al. 1992 ; GREENBERG and LOPES 1996 ; HENRY and PATTON-VOGT 1998 ). In contrast, studies now in progress suggest that the reduced phospholipid methylation in S. pombe cells grown in the presence of choline may occur at a post-transcriptional level. Future studies will be directed to elucidating these important regulatory phenomena.

	FOOTNOTES

This paper is dedicated to the memory of our colleague, Serafin Fernandez (1953–1985), who initiated this study on S. pombe choline auxotrophs at the Albert Einstein College of Medicine in the 1980s.

	ACKNOWLEDGMENTS

We are grateful to Dennis Vance for providing us with the rat liver PEMT cDNA. We also thank Paul Young for providing us with the S. pombe HindIII library. This research was supported by a National Institutes of Health grant (GM-19629 to S.A.H.). M.I. Kanipes was supported in part by a National Institutes of Health predoctoral fellowship (F31-GM-14827). This work is taken in part from a Ph.D. thesis by M.I.K.

Manuscript received September 4, 1997; Accepted for publication June 3, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALTSCHUL, S., W. GISH, W. MILLER, E. MYERS, and D. LIPMAN, 1990  Basic local alignment tool. J. Mol. Biol. 215:403-410[Medline].

ATKINSON, K. D., B. JENSEN, A. I. KOLAT, E. M. STORM, and S. A. HENRY et al., 1980a  Yeast mutants auxotrophic for choline or ethanolamine. J. Bacteriol. 141:558-564[Abstract/Free Full Text].

ATKINSON, K., S. FOGEL, and S. A. HENRY, 1980b  Yeast mutant defective in phosphatidylserine synthase. J. Biol. Chem. 255:6653-6661[Abstract/Free Full Text].

BREMER, J. and D. M. GREENBERG, 1961  Methyl transfering enzyme system of microsomes in the biosynthesis of lecithin (phosphatidylcholine). Biochim. Biophys. Acta 46:205-216.

CARMAN, G. M. and G. M. ZEIMETZ, 1996  Regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 271:13293-13296[Free Full Text].

CROCKEN, B. J. and J. F. NYC, 1964  Phospholipid variations in mutant strains of Neurospora crassa. J. Biol. Chem. 239:1727-1730[Free Full Text].

CUI, Z., J. VANCE, M. CHEN, D. VOELKER, and D. VANCE, 1993  Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase: a specific biochemical and cytological marker for a unique fraction in rat liver. J. Biol. Chem. 268:16655-16663[Abstract/Free Full Text].

CUI, Z., M. HOUWELING, and D. E. VANCE, 1995  Expression of phosphatidlyethanolamine N-methyltransferase-2 in McArdle-RH7777 hepatoma cells inhibits the CDP-choline pathway for phosphatidylcholine biosynthesis via decreased gene expression of CTP: phosphocholine cytidylyltransferase. Biochem. J. 312:939-945.

FERNANDEZ, S., M. J. HOMANN, S. A. HENRY, and G. M. CARMAN, 1986  Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe. J. Bacteriol. 166:779-786[Abstract/Free Full Text].

GREENBERG, M. L. and J. M. LOPES, 1996  Genetic regulation of phospholipid biosynthesis in yeast. Microbiol. Rev. 60:1-20[Free Full Text].

GREENBERG, M. L., L. S. KLIG, V. A. LETTS, B. S. LOEWY, and S. A. HENRY, 1983  Yeast mutant defective in phosphatidylcholine synthesis. J. Bacteriol. 153:791-799[Abstract/Free Full Text].

GRIAC, P., M. J. SWEDE, and S. A. HENRY, 1996  The role of phosphatidylcholine biosynthesis in the regulation of the INO1 gene of yeast. J. Biol. Chem. 271:25692-25698[Abstract/Free Full Text].

GUTZ, H., H. HESLOT, U. LEUPOLD and N. LOPRIENO, 1974 Schizosaccharomyces pombe, pp. 395–446 in Handbook of Genetics, edited by R. C. KING. Plenum Press, New York.

GYGAX, A. and P. THURIAUX, 1984  A revised chromosome map of the fission yeast Schizosaccharomyces pombe. Curr. Genet. 8:85-92.

HENRY, S. A., 1982 Membrane lipids of yeast: biochemical and genetic studies, pp. 101–158 in Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, edited by J. N. STRATHERN, E. W. JONES, and J. R. BROACH. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

HENRY, S. A., and J. L. PATTON-VOGT, 1998 Genetic regulation of phospholipid metabolism: yeast as a model eukaryote, pp. 133–179 in Progress in Nucleic Acid Research and Molecular Biology, Vol. 61, edited by W. E. COHN and K. MOLDAVE. Academic Press, San Diego.

HOROWITZ, N. H., 1946  The isolation and identification of a natural precursor of choline. J. Biol. Chem. 162:413-419[Free Full Text].

HOROWITZ, N. H., D. BONNER, and M. B. HOULAHAN, 1945  The utilization of choline analogues by cholineless mutants of Neurospora. J. Biol. Chem. 159:145-151[Free Full Text].

HUBBARD, S. C. and S. BRODY, 1975  Glycerophospholipid variation in choline and inositol auxotrophs of Neurospora crassa: internal compensation among zwitterionic and anionic species. J. Biol. Chem. 250:7173-7181[Abstract/Free Full Text].

INGROSSO, D., A. FOWLER, J. BLEIBAUM, and S. CLARKE, 1989  Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA and small molecule S-adenosylmethionine-dependent methyltransferases. J. Biol. Chem. 264:20131-20139[Abstract/Free Full Text].

JOHNSON, B. F., 1967  Growth of the fission yeast Schizosaccharomyces pombe with late, eccentric, lytic fission in an unbalanced medium. J. Bacteriol. 94:192-195[Abstract/Free Full Text].

KANIPES, M. I. and S. A. HENRY, 1997  The phospholipid methyltransferases in yeast. Biochim. Biophys. Acta 1348:134-141[Medline].

KENNEDY, E. P. and S. B. WEISS, 1956  The function of cytidine coenzymes in the biosynthesis of phospholipids. J. Biol. Chem. 222:193-214[Free Full Text].

KENT, C., 1995  Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem. 64:315-343[Medline].

KINNEY, A. J., 1993 Phospholipid headgroups, pp. 231–258 in Lipid Metabolism in Plants, edited by T. S. MOORE. CRC Press, Boca Raton, FL.

KODAKI, T. and S. YAMASHITA, 1987  Yeast phosphatidylethanolamine methylation pathway. J. Biol. Chem. 262:15428-15435[Abstract/Free Full Text].

KODAKI, T. and S. YAMASHITA, 1989  Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption. Eur. J. Biochem. 185:243-251[Medline].

KOHLI, J., H. HOTTINGER, P. MUNZ, A. STRAUSS, and P. THURIAUX, 1977  Genetic mapping in Schizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidization. Genetics 87:471-489[Abstract/Free Full Text].

KOVAC, L., I. GBELSKA, V. POLIACHOVA, J. SUBIK, and V. KOVACOVA, 1980  Membrane mutants: a yeast mutant with a lesion in phosphatidylserine biosynthesis. Eur. J. Biochem. 111:491-501[Medline].

KYTE, J. and R. F. DOOLITTLE, 1982  A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132[Medline].

LOEWY, B. S. and S. A. HENRY, 1984  The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes. Mol. Cell. Biol. 4:2479-2485[Abstract/Free Full Text].

MAJUMDER, A. L., M. D. JOHNSON, and S. A. HENRY, 1997  1L-myo-inositol 1-phosphate synthase. Biochim. Biophys. Acta 1348:245-256[Medline].

MARKHAM, P. and B. W. BAINBRIDGE, 1979  Characterization of a new choline locus in Aspergillus nidulans and its significance for choline metabolism. Genet. Res. 32:303-310.

MAUNDRELL, K., 1993  Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127-130[Medline].

MCGRAW, P. and S. A. HENRY, 1989  Mutations in the Saccharomyces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis. Genetics 122:317-330[Abstract/Free Full Text].

MUNZ, P., K. WOLF, J. KOHLI and U. LEUPOLD, 1989 Genetics overview, pp. 1-469 in Molecular Biology of the Fission Yeast, edited by A. NASIM, P. YOUNG and B. JOHNSON. Academic Press, Inc., San Diego.

PALTAUF, F., S. KOHLWEIN and S. A. HENRY, 1992 Regulation and compartmentalization of lipid synthesis in yeast, pp. 415–500 in The Molecular and Cellular Biology of the Yeast Saccharomyces, edited by J. BROACH, E. JONES and J. PRINGLE. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

PATTON-VOGT, J. L., P. GRIAC, A. SREENIVAS, V. BRUNO, and S. DOWD et al., 1997  Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation. J. Biol. Chem. 272:20873-20883[Abstract/Free Full Text].

PERKINS, D. D., 1949  Biochemical mutants in the smut fungus Ustilago maydis. Genetics 34:607-626[Free Full Text].

RIDGWAY, N. D. and D. E. VANCE, 1987  Purification of phosphatidylethanolamine N-methyltransferase from rat liver. J. Biol. Chem. 262:17231-17239[Abstract/Free Full Text].

ROTHSTEIN, R. J., 1983 One step gene disruption in yeast, pp. 202–211 in Methods in Enzymology: Recombinant DNA, edited by R. WU, L. GROSSMAN and K. MOLDAVE. Academic Press, New York.

SANGER, F., S. NICKLEN, and A. R. COULSEN, 1977  DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467[Abstract/Free Full Text].

SCARBOROUGH, G. A. and J. F. NYC, 1967  Methylation of ethanolamine phosphatides by microsomes from normal and mutant strains of Neurospora crassa. J. Biol. Chem. 242:238-242[Abstract/Free Full Text].

SIKORSKI, R. S. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27[Abstract/Free Full Text].

SIPICZKI, M., 1989 Taxonomy and phylogenesis, pp. 431–452 in Molecular Biology of the Fission Yeast, edited by A. NASIM, P. YOUNG and B. F. JOHNSON. Academic Press, San Diego.

STEINER, M. R. and R. L. LESTER, 1972  In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim. Biophys. Acta 260:222-243[Medline].

SUMMERS, E. F., V. A. LETTS, P. MCGRAW, and S. A. HENRY, 1988  Saccharomyces cerevisiae cho2 mutants are deficient in phospholipid methylation and cross-pathway regulation of inositol synthesis. Genetics 120:909-922[Abstract/Free Full Text].

SWEDE, M. J., K. A. HUDAK, J. M. LOPES and S. A. HENRY, 1992 Strategies for generating phospholipid synthesis mutants in yeast, pp. 21–34 in Methods in Enzymology: Phospholipid Biosynthesis, Vol. 29, edited by D. E. VANCE and E. A. DENNIS. Academic Press, Orlando, FL.

TESSITORE, L., Z. CUI, and D. E. VANCE, 1997  Transient inactivation of phosphatidylethanolamine N-methyltransferase 2 and activation of cytidine triphosphate: phosphocholine cytidyltransferase during non-neoplastic liver growth. Biochem. J. 322:151-154.

VANCE, D. E., C. WALKEY, and Z. CUI, 1997  Phosphatidylethanolamine N-methyltransferase from liver. Biochim. Biophys. Acta 1348:142-150[Medline].

WAECHTER, C. J. and R. J. LESTER, 1971  Regulation of phosphatidylcholine synthesis in Saccharomyces cerevisiae. J. Bacteriol. 105:837-843[Abstract/Free Full Text].

WRIGHT, A., K. MAUNDRELL, W. D. HEYER, D. BEACH, and P. NURSE, 1986  Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Plasmid 15:156-158[Medline].

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