Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase

Abstract

CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation.

Figure 1. Alignment of amino acid sequences of C. reinhardtii ECT and other ECTs in human, rat and yeast

The amino acid sequences of C. reinhardtii ECT (cECT; GenBank® accession number AAO60076), human ECT (hECT; NP_002852), rat ECT (rECT; NP_446020) and yeast ECT (yECT; BAA09310) are compared. The conserved HXGH motifs are indicated with asterisks, and the RTXGVSTT signature in cytidylyltransferase family is underlined. Residue numbers for amino acids are shown on the left. Gaps in alignments are indicated by dashes. Residues that are identical in three or more sequences are shaded in grey.

Figure 2. Hydropathy and α-helix amphiphilicity profiles of the predicted protein sequence of C. reinhardtii ECT

(A) Hydropathy was analysed by the method of Kyte and Doolittle [22]. Positive values represent hydrophobicity. (B) The sequence was analysed for amphipathic α-helices using the algorithm of Chou and Fasman [23]. The predicted membrane-spanning domain is indicated as TM.

Figure 3. Expression of MBP–ECT fusion gene in E. coli and its ECT activity

(A) Cell extracts from induced or uninduced bacterial cultures and purified fusion protein were subjected to SDS/PAGE. Lane 1, protein markers [molecular mass (MM) in kDa]; lane 2, cell extract from uninduced cells; lane 3, cell extract from induced cells; lane 4, purified chimaeric protein. (B) ECT enzymic assays were performed using the same cell extracts and purified fusion protein as in (A). In the enzyme assay, the reaction mixture included 30 μg of protein from E. coli cell extract or 15 μg of purified protein. The reaction products were separated by TLC as described in the Experimental section. Lane 1, substrate standards; lane 2, reaction with uninduced cell extract; lane 3, reaction with induced cell extract; lane 4, reaction with purified protein. P-Ethn, phosphoethanolamine; CDP-Ethn, CDP-ethanolamine. The activity assay of the expressed ECT in E. coli was repeated independently more than three times, and the result shown is typical of all the replicate experiments that gave the same results.

Figure 4. ECT activity of the MBP–ECT fusion protein expressed in E. coli under different conditions

The reaction mixture contained 15 μg of purified protein and was incubated at 30 °C for 30 min as described in the Experimental section. The reaction products were separated by TLC. Lane 1, no enzyme added; lane 2, with enzyme; lane 3, no Mg²⁺ added; lane 4, no CTP added; lane 5, no CDP-ethanolamine added. The data shown are from a single experiment that was repeated three times with the same results.

Figure 5. Nucleic acid analysis of the C. reinhardtii ECT gene and regulation of its transcription

(A) Southern hybridization of genomic ECT gene. Genomic DNA was digested by EcoRI (lane 1), EcoRV (lane 2), HindIII (lane 3), XmnI (lane 4), PvuII (lane 5) and PstI (lane 6), separated on a 0.8% agarose gel, transferred on to a Nytran membrane, and hybridized to a radiolabelled 0.4 kb probe from PCR of 3′ coding region of ECT cDNA. (B) Northern hybridization of total RNA from C. reinhardtii cells during reflagellation. Total cellular RNA was prepared from cells before deflagellation (pre), and during the reflagellation after pH shock at 10, 30, 60, 90 and 120 min, subjected to electrophoresis on a 1% formaldehyde/agarose gel, transferred on to a Nytran membrane and hybridized to the same probe as in the Southern blot.

Figure 6. ECT activity and expression of ECT gene during the cell cycle

Upper panel: the ECT activity during the cell cycle. The reaction mixture contained 200 μg of total protein and was incubated at 30 °C for 30 min as described. The reaction products were separated by TLC and the radioactivity of CDP-ethanolamine was measured by liquid-scintillation spectrometry of the product spot on the gel. Lower panel: Northern blot of total RNA from cells during the cell cycle. Total cellular RNA was prepared from cells during the cell cycle at 2, 6, 10, 14, 18 and 22 h, subjected to electrophoresis on a 1% formaldehyde/agarose gel, transferred on to a Nytran membrane and hybridized to the same probe as in the Southern blot. Values for the enzyme activity are the means±S.D. of three replicates of an experiment.