¹³C-carbon source labelling strategies to assess the role of FASII and <i>Tg</i>ATS1 in <i>T</i>. <i>gondii</i> membrane biogenesis.

<p><i>TgATS1</i>-iKO parasites were grown in the presence of ¹²C-glucose or U-¹³C-glucose in the presence or absence of ATc. Glucose is metabolized via the parasite glycolytic pathway into dihydroxyacetone-phosphate (DHAP) and phosphoenolpyruvate (PEP). These precursors are imported into the apicoplast via the apicoplast phosphate transporter (APT). PEP can then be transformed into acetyl-CoA via the action of the apicoplast pyruvate kinase (PK) and the pyruvate dehydrogenase complex (PDH). Acetyl-CoA serves as a substrate for the FASII pathway to generate growing acyl chains on an acyl carrier protein (ACP) scaffold. Glycerol 3-phosphate (G3P) can be generated from imported DHAP by the apicoplast glycerol 3-phosphate dehydrogenase (Gpda), or potentially by an as yet unidentified cytosolic G3P dehydrogenase (G3PDH). Both labelled and/or unlabelled G3P and FA can be used as a substrate by the apicoplast <i>Tg</i>ATS1 (glycerol 3-phosphate acyltransferase) to form lysophosphatidic acid (LPA). These LPA species can potentially be exported from the apicoplast towards the ER to be assembled into phospholipids (PL) and/or have its FA elongated via the three ER elongases (ELO). In order to assess ER elongation, <i>TgATS1</i>-iKO parasites were grown in the presence or absence of ATc together with ¹²C-acetate or U-¹³C-acetate, which is metabolized to cytosolic acetyl-CoA by acetyl-CoA synthetase (ACS). Collectively, this strategy enabled the determination of (i) apicoplast FASII-generated FA products, (ii) products assembled via <i>Tg</i>ATS1, and (iii) PL species generated via the apicoplast. Total lipid extracts from <i>TgATS1</i>-iKO parasites grown in the described conditions were analyzed by two mass spectrometry techniques. GC-MS was used for the measurement of total FA profiles and LC-MS/MS was used to determine the relative amounts of ¹³C incorporation into individual PL molecular species. In the cartoon, unlabelled and labelled moieties are shown in black and red, respectively.