Figure 5.

Evidence of channeled de novo purine synthesis in crPAICS::2×Strep-PAICS.A, schematic describing incorporation of ¹³C₃, ¹⁵N serine–derived ¹³C₂, ¹⁵N glycine (green circles) and ¹³C formate (black circles) into the DNPB intermediates and product nucleotides. Enzyme names are italicized. B, comparison of the observed AMP isotopologue distribution in crPAICS::2×Strep-PAICS (cyan bars) and crPAICS::PAICS-2×Strep (magenta bars) with natural isotopologue abundance (black bars) confirm incorporation of ¹³C₂, ¹⁵N glycine and ¹³C formate in these cell lines. Data from three independent experiments was used for paired t tests to compare the isotopologue distribution in crPAICS::2×Strep-PAICS versus crPAICS:PAICS-2×Strep. C, in crPAICS::2×Strep-PAICS, the isotopologue distribution of newly synthesized AMP (light gray bars) is significantly different from that of IMP (dark gray bars) and the pattern predicted based on the percentage ¹³C formate incorporation in the intermediate FGAR (black bars). A significantly lower fraction of the +3 isotopologue and a significantly higher fraction of +5 isotopologue, respectively, in ATP compared to IMP is a hallmark of channeled DNPB by mitochondria-associated purinosomes. D, purinosome integrity in crPAICS::2×Strep-PAICS was disrupted by trypsinization, releasing the purinosome-bound pools of all intermediates. After trypsinization, IMP and ATP show similar isotopologue distribution patterns. In (C) and (D), values from a representative experiment are shown, one-tailed paired t test was performed with values from three independent experiments; p-value: ‘ns’- not significant, ‘∗’ <0.05, ‘∗∗’ <0.01. DNPB, de novo purine biosynthesis; FGAR, phosphoribosyl-N-formylglycineamide; IMP, inosine monophosphate; PAICS, phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase.