Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase.
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.
Yang
Wenyu
W
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Mason
Catherine B
CB
Pollock
Steve V
SV
Lavezzi
Tracey
T
Moroney
James V
JV
Moore
Thomas S
TS
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002453:Cell Cycle / Q000502:physiology; D016825:Chlamydomonas reinhardtii / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D016054:DNA, Protozoan / Q000235:genetics; D048168:Escherichia coli K12 / Q000201:enzymology; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018503:Genome, Protozoan; D006801:Humans; D058619:Maltose-Binding Proteins; D008563:Membrane Lipids / Q000096:biosynthesis*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D012316:RNA Nucleotidyltransferases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017422:Sequence Analysis, DNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
D002352:Carrier Proteins; D016054:DNA, Protozoan; D058619:Maltose-Binding Proteins; D008563:Membrane Lipids; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C504174:ECT1 protein, S cerevisiae; C020126:Ethanolamine-phosphate cytidylyltransferase