Phosphatidylethanolamine and Phosphatidylcholine Biosynthesis by the Kennedy Pathway Occurs at Different Sites in Trypanosoma Brucei 

Authors:
Luce Farine  1   , Moritz Niemann  3 , André Schneider  3 , Peter Bütikofer  1             
In:
Source: Scientific Reports
Publication Date: (2015)
Issue: 18: 5
Research Area:
Parasitology
Cells used in publication:
Trypanosoma brucei
Species: unicellular
Tissue Origin: blood
Platform:
4D-Nucleofector™ X-Unit
Experiment

Stable and transient transfection of trypanosomes: Electroporation was performed in 100 µl nucleocuvettes using Lonza 4D Nucleofector System (pulse code FI-115, “Primary Cell P3” solution). GFP constructs were expressed transiently (circular plasmids were transfected into 427 strain and slides were prepared 18–24 hours after transfection) whereas HA and cmyc constructs were stably transfected into 29–13 strain (plasmids were linearized with NotI prior to transfection). Clones were obtained by limited dilution and selected in the presence of the corresponding antibiotic, 2 µ g/ml puromycin for HA and cmyc constructs, or 15 µg/ml G418 for the in situ construct.

Abstract

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.