Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae

Abstract

1. The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Sacchuromyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type.2. Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis.3. The cloned DNA was subcloned into a 2.5-kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49 379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product.4. Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N-and C-terminal regions.5. The primary translation product contains a region showing local sequence homology with nucleotidyltransfer enzymes such as DNA polymerase (Escherichiu coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.Phosphatidylcholine is a major structural component of the cellular membranes of eukaryotes. In the yeast, Sacchuromyces cerevisiue, phosphatidylcholine comprises about 40% of the total phospholipids. Several lines of evidence indicate that phosphatidylcholine is absolutely required for yeast growth [l -31. S. cerevisiae possesses two pathways for the synthesis of phosphatidylcholine, the CDPcholine pathway and the phosphatidylethanolamine methylation pathway [4-61. Due to the feasibility of genetic manipulation of the yeast, the phosphatidylcholine-synthesizing system of S. cerevisiue has been the subject of extensive genetic studies. Mutants for all of the enzymes involved in the CDPcholine pathway and the phosphatidylethanolamine methylation pathway have been isolated [l, 2, 7-91. Characterization of these mutants has provided an insight into the genetic and

Molecular Cloning of CTP: Phosphocholine Cytidylyltransferase from Plasmodium falciparum

“…falciparum CCT gene. Only two CCT genes, from yeast [37] and rat liver [38], had been cloned when this study was initiated. Our strategy for cloning the P ,fulcipurum CCT gene was to obtain a specific DNA probe rather than relying on heterologous hybridization, which was unlikely, due to the biased codon usage of l?…”

“…Tsukagoshi and his colleagues [37] have already shown that significant degrees of local similarity exist among the yeast CCT, E. coli CDP-diacylglycerol pyrophosphatase, E. coli DNA polymerase, E. coli 3-deoxy-manno-octulosonate cytidylyltransferase and T4 DNA ligase. It should be noted that all these enzymes are involved in a nucleotidyl transfer reaction.…”

“…These mammalian CCTs appeared remarkably conserved. However, only one CCT, from the lower eukaryote S. cerevisiae, has, to our knowledge, been cloned to date [37], and our l? falciparum CCT is the second cloned enzyme from lower eukaryote.…”

Direct Involvement of Phosphatidylinositol 4-Phosphate in Secretion in the Yeast Saccharomyces cerevisiae

“…Mutations in several genes (CKI1, PCT1, CPT1, KES1, BSD1, BSD2) also suppress the secretory defects of sec14 mutants (12,14). Three genes (CKI1, PCT1, CPT1) encode enzymes in a PtdCho biosynthesis pathway (61)(62)(63) and KES1 encodes an oxysterol-binding protein (64). Furthermore, Pld1p (a phospholipase D), which is activated in sec14 mutants (65), is required for these mutations to suppress sec14 defects (65,66), suggesting that the sec14 suppression by these mutations takes place only when basal PtdCho levels are decreased or phosphatidic acid levels are elevated.…”

Regulation of Phospholipid Synthesis in the Yeast cki1Δ eki1Δ Mutant Defective in the Kennedy Pathway

“…In the CDP-DAG pathway, PC is synthesized from CDP-DAG via the reactions catalyzed by PS synthase (18 -20), PS decarboxylase (21)(22)(23), PE methyltransferase (24,25), and phospholipid methyltransferase (24,26). In the CDP-choline branch of the Kennedy pathway, PC is synthesized from choline via the reactions catalyzed by choline kinase (27), phosphocholine cytidylyltransferase (28), and choline phosphotransferase (29,30). Analyses of mutations in S. cerevisiae (4,31,32), as well as in mammalian cells (33,34) indicate that the physiological role(s) of PC synthesized by the two pathways are different.…”