The essential roles of cytidine diphosphate-diacylglycerol synthase in bloodstream form Trypanosoma brucei.
Bottom Line: Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis.These studies also suggest that phosphatidylglycerol synthesized via the phosphatidylglycerol-phosphate synthase is not synthesized from CDP-DAG, as was previously thought.TbCDS was shown to localized the ER and Golgi, probably to provide CDP-DAG for the phosphatidylinositol synthases.
Affiliation: Biomedical Sciences Research Centre, School of Biology, The University of St. Andrews, The North Haugh, St. Andrews, Fife Scotland, KY16 9ST, UK.Show MeSH
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Mentions: To examine the subcellular localization of TbCDS, we used the TbCDS CKO cell line, as the viability conferred by the tetracycline induced copy of pLew 100 TbCDS‐HA confirms that the protein is correctly expressed and localized. The TbCDS‐HA protein was detected by Western blot of total protein (Fig. 3C, lane 2). The cells were stained against the HA‐tagged CDS and for ER localized BiP protein (Bangs et al., 1993; Fig. 7). The strongest signal from HA‐tagged TbCDS comes from the region between the nucleus and kinetoplast, while punctuate staining is visible throughout the cell. Since TbCDS is a membrane protein this pattern of staining indicates that it is localized to the membrane of subcellular organelles rather than the plasma membrane. Intense staining between the nucleus and kinetoplast is indicative of a Golgi localization, which has previously been shown to be the location of the downstream enzyme, PIS (Martin and Smith, 2006a) and is consistent with a requirement of CDP‐DAG for the synthesis of the bulk of PI in the cell. The perinuclear staining, and the staining throughout the cell indicate ER localization, where TbCDS could supply CDP‐DAG required for the ER localized PIS to make PI for GPI anchors.
Affiliation: Biomedical Sciences Research Centre, School of Biology, The University of St. Andrews, The North Haugh, St. Andrews, Fife Scotland, KY16 9ST, UK.