Identifier

etd-06022004-155712

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Chlamydomonas reinhardtii Dangeard does not have two major phospholipids, PS and PC. This fact renders C. reinhardtii a desirable system for investigations of the PE biosynthetic pathway and its regulation independent of PC and PS biosynthesis. The cDNA coding for ECT protein in C. reinhardtii was cloned from a cDNA library. The ECT cDNA encodes a protein of 443 amino acid residues. The ECT protein in C. reinhardtii has a repetitive internal sequence in its N- and C-terminal halves. Each repeat half of the protein has a HXGH motif, a site considered to be in the catalytic domain. The protein has a RTXGVSTT signature sequence typical of the cytidylyltransferase family. The first 70 amino acid residues appear to be a subcellular targeting signal to mitochondria. The translated product of cloned cDNA was expressed as a fusion protein with maltose-binding protein in E. coli, and was shown to have ECT activity. Northern blot analysis showed mRNA abundance is increased by reflagellation. The enzyme requires Mg2+ and pH 7.5 for maximum activity in vitro, and it appears to have a sequential reaction mechanism. The activity of the enzyme in vivo in C. reinhardtii cells changes during the cell cycle while the mRNA level does not change. A cDNA coding for EPT protein was obtained from a C. reinhardtii cDNA library. The EPT cDNA encodes a protein of 383 amino acid residues. The EPT protein has a signature sequence and a conserved region in the CDP-alcohol phosphatidyltransferase family. Very similar membrane topology was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene coding for EPT. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized PC in the transformed yeast. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine and CMP. This provides evidence that C. reinhardtii EPT is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine.

Date

2004

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Thomas Moore

DOI

10.31390/gradschool_dissertations.3136

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