Fig. 4: Preferential utilization of mitochondrially generated ¹⁵N labeled aspartic acid for channeled de novo purine synthesis.

A Schematic describing the incorporation of ¹⁵N side chain amide nitrogen of Gln (red open circles) and backbone amine nitrogen of Asp (red solid circles) into the purine ring following diffusive de novo purine biosynthesis. The first two ¹⁵N incorporations are catalyzed by the enzymes PPAT and PFAS (utilizing Gln) and the third and fourth ¹⁵N incorporations are catalyzed by the enzymes PAICS and ADSS (utilizing Asp), respectively. Note that following the hypothesis that the diffusive and the channeled DNPB utilize the same source of ¹⁵N Asp, the respective fraction ¹⁵N Asp (x) incorporation in SAICAR and AMP will be expected to be the same, thus allowing estimation of the relative abundances of the (M + 2), (M + 3), and (M + 4) isotopologues in AMP based on the value of (x) obtained from SAICAR. When the %¹⁵N Gln incorporation in the intermediates is ~100%, the (M + 2), (M + 3), and (M + 4) isotopologues in AMP show no interference from the preexisting unlabeled nucleotide pools of AMP. Fraction Isotopologue abundance observed in the DNPB intermediates B FGAR, C SAICAR, and D ADP, respectively (Additional data in Fig. S9A). D Based on the fraction ¹⁵N Asp (x) in SAICAR, the respective fractional abundance of (M + 2), (M + 3), and (M + 4) isotopologues in ATP was predicted based on the diffusive model (Eq5 Supplementary Information) and compared to the observed values. E The observed fraction ¹⁵N Asp incorporation in SAICAR and ADP, respectively. F Schematic showing the preferential ¹⁵N incorporation via Gln and Asp into the purine ring following channeled de novo purine synthesis by mitochondria-proximal purinosomes. Purinosome metabolon formation leads to channeled synthesis by restricting the diffusion of the intermediates, only allowing the end nucleotides AMP and GMP to be released in the bulk cytosol. G Schematic showing the effect of GOT2 knockdown on channeled purine synthesis. Pathway steps impacted by siRNA treatment are indicated as red inhibition arrows. As the production of Asp in mitochondria is suppressed, GOT1 mediated cytoplasmic production increases, supporting diffusive purine de novo biosynthesis, leading to accumulation of both Asp as well as the purine biosynthesis intermediates. H The relative ratio of total peak area of the substrate Asp to the purine nucleotides AMP and GMP, respectively, in GOT2 knockdown cells compared to the control non-target siRNA treated cells. I The ratio of total peak area of purine biosynthesis intermediate IMP to the end product nucleotides AMP and GMP, respectively, in GOT2 knockdown cells compared to the control non-target siRNA treated cells. (Associated supporting data in Figs. S7D, and S9B, E). B, C, D, E data from three or H, and I four independent biological replicates are shown, bar height represents data mean. *p-value < 0.05, **p-value < 0.005, ***p-value < 0.001 for a (D), (E) pairwise or (H), (I) unpaired two-ways t-test. A, F, and G prepared in Biorender https://BioRender.com/x25177d.