De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions
Fig 4
IVDH is involved in sterol biosynthesis from leucine.
(A) PCR analysis of genomic DNA isolated from the parental EATRO1125.T7T (PCL), Δivdh-C3 and Δivdh-C8 cell lines. Amplifications were performed with primers based on sequences flanking the 5’UTR and 3’UTR fragments used to target the IVDH gene depletion (black boxes) and the 5’UTR 3’-extremity (PCR product 2), 3’UTR 5’-extremity (PCR product 3) or internal sequences of the puromycin (PAC, PCR products 4 and 5) and blasticidin (BSD, PCR products 6 and 7) resistance genes. The full-length IVDH coding sequence was also PCR-amplified with specific primers sequences (PCR product 1). As expected, PCR amplification using primers derived from the IVDH gene and drug resistant genes were only observed for the parental EATRO1125.T7T and both Δivdh cell lines, respectively. (B) Western blot analysis of the EATRO1125.T7T, Δivdh-C3 and Δivdh-C8 cell lines using anti-IVDH and anti-PFR immune sera. (C) Incorporation of [14C]-labelled leucine (L-[U-14C]-leucine) and threonine (L-[U-14C]-threonine) into sterols and fatty acids of parental (PCL), Δivdh-C3 and Δivdh-C8 cell lines. [14C]-labelled fatty acid methyl esters and sterols were separated by HPTLC after transesterification and analyzed as described in the Materials and methods section. Error bars indicate mean ± SD of 3 biological replicates. Data are normalized with the parental cell (PCL) values with an arbitrary value of 100 for the PCL samples. For more detail see Fig 2 legend. Panel D shows the mitochondrial localization of IVDH by immunofluorescence. IVDH with or without the first 33 amino acids, corresponding to the putative mitochondrial-targeting motif, was fused to the N-terminus extremity of EGFP and expressed in PCF. Fluorescence associated with EGFP is shown on the left panel and ASCT was detected with an anti-ASCT immune serum (central panel). Differential interference contrast (DIC) of cells is shown to the right of each panel. Scale bar, 5 μm.