Fig 1: Proximity-biotinylation detected hotspots on mitochondria membrane.A, immunofluorescent labeling of biotinylated protein (red) upon APEX reaction in the transduced HeLa cells. There were punctate structures outside of mitochondria (green) labeled by biotin antibody under purine deficient condition (P−) compared to purine-rich condition (P+). Given purinosome formation near mitochondria increased under purine-deficient condition (P−), most likely there were purinosome proteins biotinylated during the APEX reaction. B, Western blot of total cell lysate upon APEX reaction detected by biotin antibody. Purine-rich condition (P+) and drug-treated condition (Rapa) had fewer biotinylated protein signals compared to purine deficient condition (P−). It has been proved previously that purinosome formation was interrupted under both P+ and rapamycin-treated conditions. This indicated that the number of proteins close to SLC25A13 decreased if purinosome formation was disturbed. C, the total cell lysate upon APEX reaction was purified with biotin antibody-conjugated beads, and the elutions were analyzed with western blots. The two wildtype transporters, SLC25A13 and SLC25A38, appeared to decrease under P+ and rapamycin-treated conditions, but SLC25A13-APEX2-V5 and mitochondrial abundant protein TOMM20 amount remained the same. Purinosome proteins, PPAT, ADSL, and GART, appeared to decrease significantly under P+ and rapamycin conditions compared to P−. PAICS, ATIC, and PFAS remained similar under all three conditions. HPRT served as a negative control since it is neither part of the purinosome, nor functionally related to SLC25A13. Thus, no band was detected for HPRT. D, PLA assay of mitochondrial transporters SLC25A13 and SLC25A38. Cells were grown in purine-deficient (blue column), purine-rich (red column), or rapamycin-treated conditions (green column). The PLA fluorescent images, along with the analyzed positive cell ratio, reported in the pair of SLC25A13 (V5-tag) and SLC25A38 being 87% ± 3% positive under purine-deficient condition, 62% ± 5% positive under purine-rich condition, and 75% ± 6% upon rapamycin treatment. Numbers in the parentheses indicate the total cell number analyzed under each condition. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism. The resulting p-values were reported as follows: for the PLA of SLC25A13 and SLC25A38, comparison of purine-rich to purine-deficient conditions yielded a p-value of 0.0010, and comparison of rapamycin-treated to purine-deficient conditions yielded a p-value of 0.0374. E, the schematic illustration of purinosome-mitochondrial transporter model. Purinosome proteins in initial binary complexes in the cytoplasm, move toward mitochondria and assemble into sphere-shaped condensates proximal to the mitochondrial membrane under purine-deficient conditions. Simultaneously, the mitochondrial transporters crucial for purine de novo synthesis migrate along the mitochondrial inner membrane to be adjacent to the purinosome Created with BioRender.com.