Abstract
Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.
Highlights
Phospholipid classes, usually comprising 30–50 and 20–40%, respectively, of total phospholipid [1, 2]
PE Biosynthesis in T. brucei Procyclic Forms Depends on the Kennedy Pathway—The T. brucei genome contains putative homologues for all three enzymes involved in PE biosynthesis by the Kennedy pathway
PC levels were not significantly affected by down-regulation of PE synthesis, except for a 37% decrease in cells after RNA interference (RNAi) against CEPT (Table 2). These results demonstrate that down-regulation of ethanolamine kinase (EK), ethanolamine-phosphate cytidylyltransferase (ET), and CEPT leads to a clear reduction in cellular PE levels, indicating that the Kennedy pathway represents the major route for PE synthesis in T. brucei procyclic forms
Summary
Sequence 5Ј-GCCCAAGCTTGGATCCCTGCGAAGGATGGGAGGC-3Ј 5Ј-CGTCTAGACTCGAGGCTGAGCAGGTCATTGTGG-3Ј 5Ј-GCCCAAGCTTGGATCCTCCGTACCCGCTTTATGCG-3Ј 5Ј-CGTCTAGACTCGAGCAGATAGTGCGGACCCCG-3Ј 5Ј-GCCCAAGCTTGGATCCCAACGCCATTACCGTCACG-3Ј 5Ј-CGTCTAGACTCGAGCAATCACGAACGAGCCACC-3Ј 5Ј-GCCCAAGCTTGGATCCCTTTCGCACACGCTCCACTATGAG-3Ј 5Ј-CGTCTAGACTCGAGGCGGGCAGCTAACGAATATCAGAC-3Ј brane fusion and budding events [10, 12]. A more detailed analysis revealed that both T. brucei bloodstream and procyclic forms are rich in alk-1-enyl-acyl- and alkyl-acyl-type PE species [19]. In contrast to other parasitic organisms, trypanosomes synthesize phospholipids de novo, the pathways for phospholipid synthesis in T. brucei have not been studied in great detail [20, 21]. One exception is the biosynthesis of glycosylphosphatidylinositol (GPI) lipids, where T. brucei has been used as a model organism to delineate the individual steps leading to GPI assembly [22]. We have shown that the pathways for PE and GPI synthesis in T. brucei procyclic forms are tightly connected. The importance of PE synthesis for GPI assembly and cell viability prompted us to elucidate the complete biosynthetic pathway in T. brucei procyclic forms. Knocking down any of the enzymes involved in PE synthesis affects the rate of PE synthesis and the cellular phospholipid composition in T. brucei procyclic forms
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