PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance.

To precisely define the functional sequence of the CHO1 gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the original 4.5-kilobase (kb) CHO1 clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of cho1 mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression. Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHO1 gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opi1, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When cho1 mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in cho1 cells.     

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.     

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.     

The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis.

The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study. A polypeptide of 237 amino acids was deduced from the PSS sequence. H. pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart. Expression of the putative pss gene in maxicells gave rise to a product of approximately 26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da. A manganese-dependent PSS activity was found in the membrane fractions of the E. coli cells overexpressing the H. pylori pss gene product. This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices. The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid. However, an isogenic pss gene-disrupted mutant of H. pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism.     

The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

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 (cho1Delta) 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.     

PHOSPHATIDYLSERINE SYNTHASE1 is Required for Inflorescence Meristem and Organ Development in Arabidopsis

<正>Phosphatidylserine(PS),a quantitatively minor membrane phospholipid,is involved in many biological processes besides its role in membrane structure.One PS synthesis gene,PHOSPHATIDYLSERINE SYNTHASE1(PSS1),has been discovered to be required for microspore development in Arabidopsis thaliana L.but how PSS1 affects postembryonic development is still largely unknown.Here,we show that PSS1 is also required for inflorescence meristem and organ development in Arabidopsis.Disruption of PSS1 causes severe dwarfism,smaller lateral organs and reduced size of inflorescence meristem. Morphological and molecular studies suggest that both cell division and cell elongation are affected in the pss1-1 mutant.RNA in situ hybridization and promoter GUS analysis show that expression of both WUSCHEL(WUS) and CLAVATA3(CLV3) depend on PSS1.Moreover,the defect in meristem maintenance is recovered and the expression of WUS and CLV3 are restored in the pss1-1 clv1-1 double mutant. Both SHOOTSTEMLESS(STM) and BREVIPEDICELLUS(BP) are upregulated,and auxin distribution is disrupted in rosette leaves of pss1-1.However,expression of BP,which is also a regulator of internode development,is lost in the pss1-1 inflorescence stem.Our data suggest that PSS1 plays essential roles in inflorescence meristem maintenance through the WUS-CLV pathway,and in leaf and internode development by differentially regulating the class I KNOX genes.     

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.     

Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast.

In Saccharomyces cerevisiae, amino acid permeases are divided into two classes. One class, represented by the general amino acid permease GAP1, contains permeases regulated in response to the nitrogen source. The other class, including the high affinity tryptophan permease, TAT2, consists of the so-called constitutive permeases. We show that TAT2 is regulated at the level of protein stability. In exponentially growing cells, TAT2 is in the plasma membrane and also accumulates in internal compartments of the secretory pathway. Upon nutrient deprivation or rapamycin treatment, TAT2 is transported to and degraded in the vacuole. The ubiquitination machinery and lysine residues within the NH(2)-terminal 31 amino acids of TAT2 mediate ubiquitination and degradation of the permease. Starvation-induced degradation of internal TAT2 is blocked in sec18, sec23, pep12, and vps27 mutants, but not in sec4, end4, and apg1 mutants, suggesting that, upon nutrient limitation, internal TAT2 is diverted from the late secretory pathway to the vacuolar pathway. Furthermore, our results suggest that TAT2 stability and sorting are controlled by the TOR signaling pathway, and regulated inversely to that of GAP1.     

Cloning and expression of a wheat (Triticum aestivum L.) phosphatidylserine synthase cDNA. Overexpression in plants alters the composition of phospholipids

We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.     

Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase [EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidylserine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae.     

Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.     

Yeast mutants auxotrophic for choline or ethanolamine.

Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures.     

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1Δ/Δ, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1Δ/Δ mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1Δ/Δ psd2Δ/Δ double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1Δ/Δ mutant. The psd1Δ/Δ psd2Δ/Δ mutant exhibits phenotypes similar to those of the cho1Δ/Δ mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.     

SACCHAROMYCES CEREVISIAE Recessive Suppressor That Circumvents Phosphatidylserine Deficiency

Phenotypic reversion of six independent Saccharomyces cerevisiae cho1 mutants was shown to be due predominantly to mutation of an unlinked gene, eam1. The eam1 gene was located very close to ino1 on chromosome X by meiotic tetrad analysis. Recessive eam1 mutations did not correct the primary cho1 defect in phosphatidylserine synthesis but made endogenous ethanolamine available for sustained nitrogenous phospholipid synthesis. A novel biochemical contribution to nitrogenous lipid synthesis is indicated by the eam1 mutants.     

Two Recessive Suppressors of SACCHAROMYCES CEREVISIAE CHO1 That Are Unlinked but Fall in the Same Complementation Group

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.     

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.