Fig. 2: Detection of channeling in the pyrimidine synthesis pathway.

A Schematic showing the two routes of ¹³C labeled aspartic acid (Asp) isotopologues generation from ¹³C6 Glc: by TCA cycle and pyruvate carboxylase (PC) activity. Additional data and description in Figs. S3–5 and S6A–F. Upon 6 h of labeling, the M + 2 species is generated by TCA cycle, while the (M + 3) and (M + 4) species are generated by PC activity. B Tracing the flow of labeled pyruvate and CO₂ derived atoms of oxaloacetic acid (OAA) and aspartic acid (Asp) into the pyrimidine ring. Structures of key metabolites generated from ¹³C6 Glc (additional description in Figs. S5 and S6A, B) and incorporation of labeled atoms into uridine base ring. Labeled Pyruvate, generates (M + 2), (M + 3), and (M + 4) labeled aspartic acid, finally producing labeled UMP. The three carbons in UMP derived from Asp are indicated in magenta, blue, and green colors, respectively. As shown, except the backbone carboxylate carbon of Asp, all other carbon and nitrogen atoms are incorporated into UMP. C Mathematical model to compute the fractional abundance of various intermediates and UMP isotopologues using the observed isotopologue pools of Asp. The (M + 0) and (M + 1) Asp represent unlabeled carry over, which results in (M + 5) and (M + 6) isotopologues of OMP, respectively, when combined with ¹³C5 PRPP (M + 5). The (M + 2), (M + 3), and (M + 4) isotopologues of Asp result in (M + 7), (M + 8), and (M + 9) isotopologues of OMP, respectively. OMP hence produced is converted to UMP by the enzyme UMP synthase, releasing a CO₂ molecule. Next to each isotopologue, their respective fractional abundance is shown in brackets. The combined pool of the indicated isotopologues is equated to 100%. The model integrates the scheme in (B) and the observed isotopologue fractions of the pyrimidine biosynthesis substrate Asp to predict the fractional abundance of isotopologues in the intermediates and the end product UMP originating from this pool using Eqs. 1–4 (Supplementary Information). D Comparison of the isotopologue patterns in Asp and the various pyrimidine synthesis intermediates. E Similar isotopologue patterns in Asp and the pyrimidine synthesis intermediates suggest diffusive synthesis of the intermediates from the bulk cytosolic Asp pool. This mechanism accumulates the intermediates and hence has low end product generation. F Application of the diffusive mode described in C to predict the isotopologue pattern in UMP based on that observed in orotate. G The observed (M + 6), (M + 7), and (M + 8) isotopologue fractional abundance in UTP as compared to the respective values predicted by the diffusive model described in C. H The results in G support a mitochondrial-Asp dependent channeled mechanism for the synthesis of uridine nucleotide by a pyrimidinosome complex. Such a complex would be comprised of the enzymes CAD, DHODH, and UMPS, preferentially utilize the mitochondrially generated Asp, and restrict equilibration of intermediates with their respective bulk cytosolic pools. D and G data from three independent biological replicates are shown, bar heights represent data mean. Pairwise comparison using two-way Student’s t-test was performed in G. ***p-value < 0.001. Enzyme names are italicized, and the Asp, malate, and pyruvate mitochondrial transporters are shown in magenta, skyblue, and light-green, respectively. Cit citrate, Asp aspartic acid, Pyr pyruvate, OAA oxaloacetic acid, α-KG α-ketoglutarate, Glu glutamate, UMP uridine monophosphate, UTP uridine triphosphate, AcCoA acetyl CoA. A–C, E, and H were prepared in Biorender and Chemdraw structures were used in B https://BioRender.com/x25177d.