Figure 5.

Lipid biosynthesis proceeded in Fe-S–deficient cells despite lack of cell growth. A, ISCU2^D71A cells were plated at a higher initial seeding density to obtain comparable amounts of cells at the end of the experiment, and cell proliferation was monitored by real-time imaging (n = 6, mean ± S.D. (error bars)). B, analysis of cell proliferation during the [¹³C₆]glucose tracer experiment demonstrated that growth of cells was greatly diminished after 12 h of doxycycline-induced induction of ISCU2^D71A expression (n = 6, mean ± S.D.). C, representative lipid HSQC NMR spectra from [¹³C₆]glucose-labeled cells expressing empty vector (black trace) or ISCU^D71A (blue trace). Unlabeled empty vector cells (gray) grown in the presence of unlabeled [¹²C]glucose are also shown for reference. These spectra reflect the combined resonances from all extracted lipid species in the sample. D, a glycerolipid structure is shown for partial HSQC NMR peak assignment. Side chain R can include a third acyl chain (triacylglycerol), a proton (diacylglycerol) phosphatidylcholine, phosphatidylethanolamine, etc. E, ¹³C enrichment of the various lipid functional groups was assessed by ¹H-¹³C HSQC NMR. Integrated peak areas were normalized to the methanol solvent peak and plotted as a percentage of control (empty vector) peak areas (n = 3, mean ± S.D.). *, p < 0.05.