The Plasmodium falciparum PfGatp is an Endoplasmic Reticulum Membrane Protein Important for the Initial Step of Malarial Glycerolipid Synthesis*

During its 48-h asexual life cycle within human erythrocytes, Plasmodium falciparum grows to many times its own size and divides to produce 16–32 new parasites. This rapid multiplication requires active synthesis of new membranes and is fueled by phospholipid precursors and fatty acids that are scavenged from the human host. Plasmodium membrane biogenesis relies heavily on the expression of parasite enzymes that incorporate these precursors into phospholipids. However, little is known about the genes involved in membrane biogenesis or where this process takes place within the parasite. Here, we describe the analysis in P. falciparum of the first step of phospholipid biosynthesis that controls acylation of glycerol 3-phosphate (GPAT) at the sn-1 position. We show that this activity is of parasite origin and is specific for glycerol 3-phosphate substrate. We have identified the gene, PfGAT, encoding this activity in P. falciparum and reconstituted its codon composition for optimal expression in the yeast Saccharomyces cerevisiae. PfGAT complements the lethality of a yeast double mutant gat1Δgat2Δ, lacking GPAT activity. Biochemical analysis revealed that PfGatp is a low affinity GPAT enzyme with a high specificity for C16:0 and C16:1 substrates. PfGatp is an integral membrane protein of the endoplasmic reticulum expressed throughout the intraerythrocytic life cycle of the parasite but induced mainly at the trophozoite stage. This study, which describes the first protozoan GPAT gene, reveals an important role for the endoplasmic reticulum in the initial step of Plasmodium membrane biogenesis.

[1]  I. Sherman,et al.  Biochemistry of Plasmodium (malarial parasites). , 1979, Microbiological reviews.

[2]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[3]  G. Daum,et al.  Triacylglycerol biosynthesis in yeast , 2003, Applied Microbiology and Biotechnology.

[4]  K. Kurokawa,et al.  Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy , 2002, The Journal of experimental medicine.

[5]  D. C. Gomes,et al.  Description of the adult form of Nybelinia (Syngenes) rougetcampanae Dollfus, 1960 and some new data on n. (n.) bisculata (Linton, 1889) (Trypanorhyncha: Tentaculariidae) , 1992 .

[6]  G. Carman,et al.  Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae. , 1987, The Journal of biological chemistry.

[7]  Nirbhay Kumar,et al.  Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family. , 1991, Molecular and biochemical parasitology.

[8]  Jonathan E. Allen,et al.  Genome sequence of the human malaria parasite Plasmodium falciparum , 2002, Nature.

[9]  S. Prigge,et al.  The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII. , 2003, Biochemistry.

[10]  W. Trager,et al.  Human malaria parasites in continuous culture. , 1976, Science.

[11]  T. M. Lewin,et al.  Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. , 1999, Biochemistry.

[12]  D. Goldberg,et al.  Localization of the Plasmodium falciparumPfNT1 Nucleoside Transporter to the Parasite Plasma Membrane* , 2001, The Journal of Biological Chemistry.

[13]  H. Vial,et al.  Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine , 1986, FEBS letters.

[14]  D. Goldberg,et al.  Isolation and Functional Characterization of the PfNT1 Nucleoside Transporter Gene from Plasmodium falciparum * , 2000, The Journal of Biological Chemistry.

[15]  G. Carman,et al.  Isolation and Characterization of the Saccharomyces cerevisiae DPP1 Gene Encoding Diacylglycerol Pyrophosphate Phosphatase* , 1998, The Journal of Biological Chemistry.

[16]  H. Vial,et al.  Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific marker in malarial fractionation studies. , 1986, Biochimica et biophysica acta.

[17]  Irwin W. Sherman,et al.  Malaria : parasite biology, pathogenesis, and protection , 1998 .

[18]  F. Cohen,et al.  Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray , 2003, Genome Biology.

[19]  T. M. Lewin,et al.  Physiological and nutritional regulation of enzymes of triacylglycerol synthesis. , 2000, Annual review of nutrition.

[20]  Christopher J. Tonkin,et al.  Dissecting Apicoplast Targeting in the Malaria Parasite Plasmodium falciparum , 2003, Science.

[21]  K. Athenstaedt,et al.  Phosphatidic acid, a key intermediate in lipid metabolism. , 1999, European journal of biochemistry.

[22]  J. Zou,et al.  The Initial Step of the Glycerolipid Pathway , 2001, The Journal of Biological Chemistry.

[23]  R. Bell,et al.  Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene. , 1986, The Journal of biological chemistry.

[24]  G. Holz,et al.  Lipids and the malarial parasite. , 1977, Bulletin of the World Health Organization.

[25]  K. Christiansen Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae). , 1978, Biochimica et biophysica acta.

[26]  H. Vial,et al.  Biosynthesis and dynamics of lipids in Plasmodium-infected mature mammalian erythrocytes. , 1990, Blood cells.

[27]  C. McMaster,et al.  Differential Partitioning of Lipids Metabolized by Separate Yeast Glycerol-3-phosphate Acyltransferases Reveals That Phospholipase D Generation of Phosphatidic Acid Mediates Sensitivity to Choline-containing Lysolipids and Drugs* , 2002, The Journal of Biological Chemistry.

[28]  T. Mitamura,et al.  Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum. , 2000, Parasitology international.

[29]  G. McFadden,et al.  The apicoplast: a plastid in Plasmodium falciparum and other Apicomplexan parasites. , 2003, International review of cytology.

[30]  G. Daum,et al.  Synthesis of Triacylglycerols by the Acyl-Coenzyme A:Diacyl-Glycerol Acyltransferase Dga1p in Lipid Particles of the Yeast Saccharomyces cerevisiae , 2002, Journal of bacteriology.

[31]  H. Sul,et al.  Acyltransferases of de novo glycerophospholipid biosynthesis. , 1999, Progress in lipid research.

[32]  L. Hyman,et al.  Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae. , 1998, Nucleic acids research.

[33]  R. Lehner,et al.  Biosynthesis of triacylglycerols. , 1996, Progress in lipid research.

[34]  H. Vial,et al.  A Class of Potent Antimalarials and Their Specific Accumulation in Infected Erythrocytes , 2002, Science.

[35]  R. Coleman,et al.  Enzymes of glycerolipid synthesis in eukaryotes. , 1980, Annual review of biochemistry.

[36]  K. Haldar,et al.  Identification and localization of ERD2 in the malaria parasite Plasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi. , 1993, The EMBO journal.

[37]  Patricia De la Vega,et al.  Discovery of Gene Function by Expression Profiling of the Malaria Parasite Life Cycle , 2003, Science.

[38]  G. Carman,et al.  Isolation and Characterization of the Saccharomyces cerevisiae LPP1 Gene Encoding a Mg2+-independent Phosphatidate Phosphatase* , 1998, The Journal of Biological Chemistry.

[39]  T. Mitamura,et al.  Lipid metabolism in Plasmodium falciparum-infected erythrocytes: possible new targets for malaria chemotherapy. , 2003, Microbes and infection.

[40]  D. Roos,et al.  Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  K. Athenstaedt,et al.  Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae , 1999, Journal of bacteriology.

[42]  D. Goldberg,et al.  Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1β and inhibits its PKC‐mediated nucleotide exchange activity in vitro , 2001, Molecular microbiology.