
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02121-5
10.1016/j.jbc.2024.107620
107620
Research Article
Purinosomes spatially co-localize with mitochondrial transporters
Sha Zhou
Benkovic Stephen J. sjb1@psu.edu
∗
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA
∗ For correspondence: Stephen J. Benkovic sjb1@psu.edu
02 8 2024
9 2024
02 8 2024
300 9 10762022 2 2024
26 6 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.

Keywords

purinosomes
de novo purine biosynthesis
mitochondrial transporter
proximity ligation assay (PLA)
APEX
Abbreviations

ADSL adenylosuccinate lyase

AICAR aminoimidazole-4-carboxamide ribonucleotide

AIR aminoimidazole ribonucleotide

APEX engineered ascorbic acid peroxidase

ATIC bifunctional enzyme AICAR Tfase/ IMP cyclohydrolase

ATP adenosine triphosphate

CAIRS phosphoribosyl aminoimidazole carboxylase

DMEM medium Dulbecco's Modified Eagle Medium

EGFP enhanced green fluorescent protein

FBS fetal bovine serum

FGAM N-formylglycinamidine ribonucleotide

FGAR N-formylglycinamide ribonucleotide

GAR Tfase phosphoribosylglycinamide formyltransferase

GARS phosphoribosylglycinamide synthetase

HPRT hypoxanthine phosphoribosyltransferase

IMP inosine monophosphate

PAICS phosphoribosyl aminoimidazole succinocarboxamide synthetase

PFAS phosphoribosyl formylglycinamidine synthase

PRA 5-phosphoribosylamine

PPAT PRPP amidotransferase

PRPP phosphoribosyl pyrophosphate

SAICAR N-succinocarboxyamide-5-aminoimidazole ribonucleotide

PLA proximity ligation assay

SLC25A13 electrogenic aspartate/glutamate antiporte, mitochondrial

TOMM20 mitochondrial import receptor subunit TOM20 homolog

V5 tag a short peptide tag for detection and purification of proteins, sequence is GKPIPNPLLGLDST

Reviewed by members of the JBC Editorial Board. Edited by Craig Cameron
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pmcPurine homeostasis in mammalian cells is intricately regulated by the coordinated interplay of complementary salvage and de novo biosynthetic pathways. Under normal physiological conditions, the salvage pathway maintains purine nucleotide levels, whereas the de novo pathway is upregulated in response to elevated purine demand (1, 2, 3). Central to this regulation are purinosomes, dynamic assemblies that enhance metabolic flux through the de novo pathway by preventing the loss of unstable intermediates through channeling. These structures exhibit reversible formation in response to purine depletion, and the percentage of cells containing purinosomes positively correlates with the extent of purine salvage deficiency observed previously (4, 5, 6, 7).

The de novo purine biosynthetic pathway is a complex series of energy-intensive processes that results in the synthesis of inosine monophosphate (IMP) from phosphoribosyl pyrophosphate (PRPP) through 10 sequential reactions catalyzed by six enzymes (8, 9, 10). The pathway begins with the conversion of PRPP to 5-phosphoribosylamine (PRA) by PRPP amidotransferase (PPAT). Subsequently, PRA transforms N-formylglycinamide ribonucleotide (FGAR), involving glycyl and formyl group addition. This step is catalyzed by the phosphoribosylglycinamide synthetase (GARS) and phosphoribosylglycinamide formyltransferase (GAR Tfase) domains of GART. Continuing along the pathway, FGAR is further processed into N-formylglycinamidine ribonucleotide (FGAM) by phosphoribosyl formylglycinamidine synthase (PFAS), and the third domain of GART facilitates the conversion of FGAM into aminoimidazole ribonucleotide (AIR). The bifunctional enzyme phosphoribosyl aminoimidazole carboxylase (CAIRS)/phosphoribosyl aminoimidazole succinocarboxamide synthetase (PAICS) then converts AIR into N-succinocarboxyamide-5-aminoimidazole ribonucleotide (SAICAR) through a two-step reaction. Adenylosuccinate lyase (ADSL) mediates the conversion of SAICAR to aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is further processed into IMP with the assistance of the bifunctional enzyme AICAR Tfase/IMP cyclohydrolase (ATIC). The enzymes within the de novo pathway rely on diverse amino acid substrates and cofactors. Each step of IMP synthesis demands five molecules of ATP, two molecules each of glutamine and formate, and one molecule each of glycine, aspartate, and carbon dioxide. Tricarboxylic acid cycle intermediates serve as sources for glutamine and aspartate, while glycine is a byproduct of serine-driven one-carbon metabolism. Formate, exported from mitochondria, plays a pivotal role in the biosynthesis of the 10-formyltetrahydrofolate cofactor, crucial for the activities of GART and ATIC (11).

Purinosomes, intricate high-ATP-consuming complexes that exhibit the properties of liquid condensates, exhibit co-localization with mitochondria, fostering a close spatial relationship. In a prior investigation, purinosomes were represented by transfected PFAS-EGFP punctuated structures, as identified through high-resolution confocal microscopy (5, 12, 13). The outcome unveiled a specific spatial alignment of purinosomes along the mitochondria. This observed co-localization implied a reciprocal relationship, indicating mutual benefits for both purinosomes and mitochondria. A metabolic tracing study revealed that purinosomes directly uptake glycine from mitochondria instead of cytoplasm (6). The coexistence and alignment of these structures likely contribute to the efficient utilization of energy resources and metabolic intermediates, thereby enhancing cellular processes (Fig. 1A).Figure 1 Schematic illustration of purine de novo synthesis pathway detected using proximity labeling assay (PLA) and proximity labeling (APEX).A, the scheme of de novo purine biosynthesis with mitochondrial transporters supplied aspartate, glutamate, and glycine. Purinosome is physically co-localized with cell mitochondria such that it can directly uptake glutamate for the first and fourth reaction, glycine for the second reaction, and aspartate for the seventh reaction in the pathway. B, schematic illustration of proximity labeling assay (PLA) used to detect purinosome proteins co-localization with mitochondrial transporters. C, schematic illustration of protein proximity labeling performed via mitochondrial transporter SLC25A13 conjugated to APEX2. Created with BioRender.com.

Mitochondria have a double-membrane system, comprising inner and outer membranes with an intermembrane space between them (14). The outer membrane is permeable to numerous small molecules, electrolytes, and protons (15, 16). Thus, the focus on studying small molecule transport primarily centers on inner membrane transporters. Mitochondrial transporters, the SLC25 family, are nuclear-coded proteins located in the inner mitochondrial membrane. They facilitate the transport of diverse substrates crucial for cellular metabolism, contributing to processes like oxidative phosphorylation, the tricarboxylic acid cycle, and fatty acid metabolism. Their specificity in transporting nucleotides, amino acids, cofactors, and ions supports cellular homeostasis with tight regulation responsive to factors like substrate availability and energy status (17, 18). Given the importance of glutamate, aspartate, and formate in the purine de novo synthesis pathway, our study focused on mitochondrial transporters related to these molecules. The two isoforms, SLC25A13 (AGC2) and SLC25A12 (AGC1) are the prominent mitochondrial transporters in the SLC25 family, confirmed to transport aspartate/glutamate through the inner membrane. Operating as a vital component in the urea cycle and myelin synthesis, these transporters exchange glutamate with a proton for aspartate. Both exhibit a unique three-domain structure: an N-terminal domain containing eight calcium-regulated EF hands, a carrier domain, and a C-terminal amphipathic helix. Their functional structure, validated as a dimer, is noteworthy within the SLC25 family (19, 20, 21, 22) Research on SLC25A38 has been limited, yet it was reported to transport glycine through the inner membrane and to play an important role in heme biosynthesis (23). The transportation of formate through the mitochondrial inner membrane remains an unresolved aspect.

In the investigation of microscale protein complex co-localization within cells, several methods have emerged, and one such approach involves in vivo proximity labeling to identify endogenous protein co-localization. In this method, antibodies sourced from different species are employed to recognize two endogenous proteins in fixed cells. Subsequently, secondary antibodies with conjugated nucleotides are utilized; these nucleotides can be ligated into a circular DNA structure if the two proteins are within a 40 nm distance. The circular DNA is then amplified using fluorescently labeled nucleotides, resulting in detectable punctuated structures under microscopes. The presence of fluorescent puncta, without modifying the cell genome, indicates the co-localization of the two proteins (24). In our study, we employed the PLA assay to discern the proximity localization between purinosome proteins and mitochondrial transporters (Fig. 1B).

An alternative method for detecting protein co-localization is APEX (Engineered Ascorbic Acid Peroxidase). APEX2 is designed to comprehensively capture the location, timing, and interactions of a target protein within living cells (25). We genetically fused APEX2 to the mitochondrial transporter SLC25A13 and stably expressed the fused construct in cells in this study. Biotin phenol was used as a substrate, which, in the presence of hydrogen peroxide, could be converted into a biotin-phenoxyl radical by APEX2. This radical covalently binds to nearby amino acids, such as tyrosine, within a short radius (typically within 20 nm) around the APEX2-fused mitochondrial transporter due to the limited exposure time of hydrogen peroxide and the short-lived availability of the biotin-phenoxyl radical (25, 26). Subsequently, cells were lysed, and biotinylated proteins were purified using a biotin antibody. The analysis of purified proteins yielded insights into protein-protein interactions and spatial organization near the mitochondrial transporter in live cells (Fig. 1C).

With these two methods, we found that the SLC25A13 and SLC25A38 mitochondrial transporters are proximal to proteins found within the de novo purine biosynthetic pathway. Strong PLA signals were detected between the transporters and GART and ADSL. With the APEX2 fused transporter, we observed biotinylation of all six pathway enzymes confirming that an intact purinosome was adjacent to the transporters. This finding suggests that the required amino acid cofactors likewise are channeled to the relevant pathway enzymes.

Results

Proximity ligation assay (PLA) detected purinosome proteins co-localized with mitochondrial transporters that provide precursors for purine de novo synthesis

We first investigated the spatial relationship between purinosome proteins and mitochondrial transporters at the endogenous level. The proximity ligation assay (PLA) employed in this study was an antibody-based technique conducted on fixed cells. It necessitated the simultaneous binding of two endogenous proteins by antibodies from distinct species. We utilized antibodies from rabbit and mouse origins in this study. Subsequently, the nucleotide-conjugated secondary antibodies bind to the primary antibodies. Amplification of DNA nucleotides on the probes occurred only when a pair of secondary antibodies were within the required proximity (less than 40 nm). This amplification generated a fluorescent signal visible as puncta foci under imaging (24). DAPI was utilized as a cell indicator, with cells containing more than six puncta foci classified as positive, and the ratio of positive cells to total cells counted was reported as the "percentage of positive cells."

Glycine, aspartate, glutamate, and formate are indispensable for purine de novo synthesis (11). Given the absence of a known mitochondrial formate transporter, we focused on the glycine transporter SLC25A38, as well as the aspartate/glutamate transporter SLC25A13. Among the four purinosome proteins requiring essential supplies from mitochondria, namely PPAT, GART, PFAS, and PAICS; PPAT and PFAS rely on glutamate, PAICS on aspartate, and GART on glycine during purine de novo synthesis. Due to antibody availability, we selected GART for our investigation. HeLa cells were cultured under purine-rich or purine-deficient conditions for 24 h prior to fixation. Previous research indicated the presence of purinosomes in HeLa cells, and the absence of purine in the medium was known to enhance purinosome formation (2, 3, 27). Additionally, rapamycin was introduced in purine-deficient conditions to disrupt purinosome integrity and disassociate the purinosome from mitochondria (12, 28). Our observations revealed that 87% ± 9% of cells exhibited positive co-localization of SLC25A38 and GART under purine-rich conditions. The PLA signal for SLC25A38 with GART under purine-deficient conditions remained at 88% ± 8%, similar to the purine-rich conditions. Subsequent rapamycin treatment resulted in 63% ± 12% of cells displaying GART and SLC25A38 colocalization, indicating the necessity for GART to remain in proximity to the mitochondrial glycine transporter (Fig. 2A). We also assessed the co-localization of GART with the aspartate/glutamate transporter SLC25A13. 57% ± 7% of cells displayed co-localization of GART and SLC25A13 under purine-rich conditions, while 87% ± 17% exhibited such co-localization under purine-deficient conditions. Even after rapamycin treatment, 76% ± 13% of cells remained positive for GART-SLC25A13 PLA (Fig. 2B). We used siRNA to knock down the expression of SLC25A38 in HeLa cells, and the expression level was confirmed with Western blot (Fig. S1A). 25% ± 13% of cells exhibited GART and SLC25A13 co-localization upon SLC25A38 knockdown, compared to 83% ± 1% of wild-type cells (Fig. S1B). Notably, the detection distance of the PLA assay was 40 nm, and considering the dimensions of mitochondria being 200 nm to 1 μm in diameter and lengths between 1 to 10 μm, the GART-SLC25A13 PLA pair could potentially cover one-20th to one-fifth of the mitochondria area, increasing the likelihood of cells being determined as GART-SLC25A13 PLA positive as long as GART was in proximity to mitochondria.Figure 2 Purinosome co-localized with mitochondrial transporters proximity labeling assay (PLA) was antibody-based to test two protein co-localization situations specifically. When the two target proteins were close enough (less than 40 nm), the PLA signal appeared in red, and cells were also dyed with DAPI (blue). If there were more than 6 PLA puncta in one cell, the cell was counted as a “positive cell”, and the percentage of positive cells to total cells was reported as instructed by the manufacturer. Cells were grown in purine-deficient (blue column), purine-rich (red column), or rapamycin-treated conditions (green column). Numbers in the parentheses indicated the total cell number analyzed under each condition. The PLA fluorescent images, along with the analyzed positive cell ratio, reported in the pair of (A) GART and SLC25A38 being 88% ± 8% positive under purine-deficient condition, 87% ± 9% positive under purine-rich condition, and 63% ± 12% upon rapamycin treatment, (B) GART and SLC25A13 being 87% ± 17% positive under purine-deficient condition, 57% ± 7% positive under purine-rich condition, and 76% ± 13% upon rapamycin treatment, (C) ADSL and SLC25A38 being 78% ± 8% positive under purine-deficient condition, 5% ± 5% positive under purine-rich condition, and 40% ± 4% upon rapamycin treatment, (D) ADSL and SLC25A13 being 58% ± 10% positive under purine-deficient condition, 9% ± 10% positive under purine-rich condition, and 23% ± 7% upon rapamycin treatment. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism. The resulting p-values were reported as follows: (A) for the PLA of GART and SLC25A38, comparison of purine-rich to purine-deficient conditions yielded a p-value of 0.9540, and comparison of rapamycin-treated to purine-deficient conditions yielded a p-value of 0.0048. (B) for the PLA of GART and SLC25A13, comparison of purine-rich to purine-deficient conditions yielded a p-value of 0.0505, and comparison of rapamycin-treated to purine-deficient conditions yielded a p-value of 0.4844. (C) for the PLA of ADSL and SLC25A38, a comparison of purine-rich to purine-deficient conditions yielded a p-value less than 0.0001, and a comparison of rapamycin-treated to purine-deficient conditions yielded a p-value of 0.005. (D) for the PLA of ADSL and SLC25A13, a comparison of purine-rich to purine-deficient conditions yielded a p-value of 0.0011, and a comparison of rapamycin-treated to purine-deficient conditions yielded a p-value of 0.0061.

In contrast, 78% ± 8% of cells showed ADSL and SLC25A38 co-localization under purine-deficient conditions, a significantly higher percentage than the 5% ± 5% PLA signal observed under purine-rich conditions. Rapamycin treatment notably reduced ADSL and SLC25A38 co-localization, leaving only 40% ± 4% of cells positive (Fig. 2C). A similar trend was observed in the co-localization of ADSL with SLC25A13. 58% ± 10% of cells exhibited positivity for the ADSL-SLC25A13 pair under purine-deficient conditions, compared to 9% ± 10% under purine-rich conditions and 23% ± 7% upon rapamycin treatment (Fig. 2D). Upon SLC25A38 knockdown, 13% ± 15% of cells displayed SLC25A13 and ADSL co-localization, whereas there were 72% ± 7% of wild-type cells being positive under purine-deficient conditions (Fig. S1C). Since ADSL does not directly uptake intermediates from mitochondrial transporters, the increased physical proximity of ADSL to mitochondrial transporters under purine-deficient conditions, coinciding with heightened purinosome generation as demonstrated in prior studies, suggests that ADSL being in proximity to mitochondria reflects the formation of intact purinosomes. Glycine is the substrate utilized in the second step of channeled purine de novo synthesis, where GART converts 5-PRA and glycine into GAR. The mitochondrial glycine supply was cut off due to the knockdown of transporter SLC25A38. Sequentially, ADSL, a later protein in the channeled synthesis pathway, disassociated from the mitochondrial transporter. We suspect the disassociation reflects the disassembly of active purinosome. Thus, the colocalization of PFAS and ADSL was checked.

PFAS performs the fourth step, while ADSL executes the eighth step of channeled purine de novo synthesis. Neither of them utilizes glycine, and they do not involve direct release-uptake of intermediates. The PFAS and ADSL combination represents the intact purinosome (29). In purine-deficient conditions, 71% ± 6% of cells were positive for a PFAS-ADSL PLA signal, compared to 43% ± 6% in purine-rich conditions. Following SLC25A38 knockdown, only 28% ± 15% of cells retained positivity under purine-deficient conditions. Through the depletion of mitochondrial glycine supply, the ratio of purinosome-containing cells under purine-deficient conditions resembled that of purine-rich conditions, signifying the absence of intact functional purinosome (Fig. S1D). The residual PLA signal between the PFAS and ADSL is consistent with earlier BiFC studies that found PFAS-ADSL complexes under purine-positive conditions but were not capable of enhanced purine synthesis (27).

We choose an additional mitochondrial transporter to examine the specificity of spatial localization of purinosome proteins with mitochondrial transporters as a negative control. Specifically, SLC25A19 serves as the thiamine diphosphate transporter and is positioned on the mitochondrial inner membrane. De novo synthesis of purines neither involves nor produces thiamine diphosphate. The proximity ligation assay (PLA) of SLC25A19 with GART revealed 25% ± 5% positivity in purine-deficient environments and 20% ± 4% in purine-rich conditions (Fig. S2A). The PLA output between SLC25A19 and ADSL indicated 10% ± 6% under purine-deficient conditions and 8% ± 5% under purine-rich conditions (Fig. S2B). The colocalization of the mitochondrial thiamine diphosphate transporter with purinosome proteins was minimal. Furthermore, there was no discernible purine-dependent impact. In summary, we hypothesized that the selective affiliation of purinosome in spatial proximity to mitochondrial transporters represents a distinctive phenomenon. To gain further insights, we employed an untargeted proximity labeling approach to elucidate the protein interactions in the vicinity of the purinosome–mitochondria interface.

Introduced APEX in the mitochondrial intermembrane space (SLC25A13-APEX2-V5)

We developed an SLC25A13-APEX construct to label nearby proteins in HeLa cells. APEX2 protein was fused to the C-termini of the mitochondrial transporter SLC25A13, with a V5 tag added to the C-termini of APEX2 (Fig. 3A). The resulting SLC25A13-APEX2-V5 construct was introduced into HeLa cells via lentiviral transduction. The expression of the protein was assessed by western blotting using SLC25A13 and V5 antibodies separately. Compared to endogenous SLC25A13, SLC25A13-APEX2-V5 exhibited a higher molecular weight but a lower expression level. The protein bands detected by both SLC25A13 and V5 antibodies displayed the same molecular weight, confirming the correct expression of SLC25A13-APEX2-V5 in transduced HeLa cells (Fig. 3B). The localization of SLC25A13-APEX2-V5 was validated by fluorescent immunostaining of fixed HeLa cells. The construct was detected using the V5 antibody, while mitochondria were labeled using the TOMM20 antibody. The overlapping colors from the V5 and TOMM20 signals indicated the proper insertion of the APEX2-conjugated SLC25A13 transporter into mitochondria (Fig. 3C).Figure 3 A HeLa cell line stablely expressing SLC25A13-APEX-V5 was generated for proximity labeling.A, schematic illustration of APEX2 conjugated mitochondrial transporter SLC25A13 with a V5 tag. The APEX2 and V5 tags were inserted on the C-terminal of SLC25A13 and transduced into HeLa mitochondrial intermembrane space. Created with BioRender.com. B, Western blot of wildtype SLC25A13 and the SLC25A13-APEX2-V5 detected by both SLC25A13 and V5 antibodies in the transduced HeLa cells. Beta-actin was used as a loading control. C, immunofluorescent labeling of V5 tag (green) and mitochondrial membrane protein TOMM20 (red) in the transduced HeLa cell proved that the SLC25A13-APEX2-V5 construct was properly expressed on mitochondria. DAPI is used to indicate cell nucleus.

Proximity-biotinylated protein purification revealed that purinosome and mitochondrial transporters formed a hotspot on mitochondria

Half of the SLC25A13 transporter resides within the mitochondrial inner membrane, while its C-termini fold is within the intermembrane area, with a size of 5 nm by 9 nm. The diameter was measured using the AI model Q9UJS0 for SLC25A13 (30). The APEX2 protein is 5 nm by 4.5 nm (APX PDB: 1V0H) (31). Consequently, a 10nm-sized structure is conjugated to the mitochondrial inner membrane, extending toward the outer membrane. The average distance between the mitochondrial outer and inner membranes is approximately 20 nm (32, 33, 34) The final distance from SLC25A13-APEX2-V5 to both mitochondrial inner and outer membranes should be around 10 nm. Following incubation with biotin-phenol and the APEX reaction, proteins proximate to SLC25A13-APEX2-V5 (within 20 nm) are biotinylated. Subsequently, fixed HeLa cells were subjected to fluorescent immunostaining, with biotinylated proteins visualized using a biotin antibody and TOMM20 serving as a mitochondrial marker for contrast. The biotin signal was observed near the vicinity of mitochondria, as anticipated. Interestingly, cells cultured in a purine-deficient medium exhibited punctuated biotinylated foci adjacent to mitochondria, while those in a purine-rich medium displayed fewer biotinylated puncta outside of mitochondria (Fig. 4A). This observation suggests a potential correlation between the puncta and purinosome formation, prompting us to further investigate through purification of biotinylated proteins from cell lysate.Figure 4 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.

Transduced HeLa cells were cultured under purine-rich, purine-deficient, and rapamycin-treated purine-deficient conditions. The APEX reaction was conducted with an equal amount of total protein from each condition that was separated using SDS-PAGE gel and transferred to a PVDF membrane. The expression levels of purinosome-related proteins, including mitochondrial transporters (SLC25A38 and SLC25A13) and purinosome proteins (PPAT, ADSL, GART, PAICS, ATIC, and PFAS), remained constant across the three culturing conditions. TOMM20, beta-actin, and HPRT served as loading controls due to their abundance, although unrelated to purine de novo synthesis. Their expression levels also remained unchanged (Fig. S3A). Biotinylated proteins were probed with a biotin antibody, revealing that biotinylated proteins following the APEX reaction were most abundant in the purine-deficient sample, followed by the rapamycin-treated sample, with the purine-rich sample exhibiting the least abundance. This result suggests an accumulation of proteins near mitochondrial SLC25A13 transporters under purine-deficient conditions, with rapamycin potentially dissociating some proteins from the transporter. Conversely, proteins were less abundant near SLC25A13 under purine-rich conditions (Fig. 4B).

To delve deeper into the status of purinosome-related proteins following the APEX reaction, we harvested cells and purified biotinylated proteins from total cell lysate using biotin antibody-conjugated agarose beads. The eluted biotinylated proteins were analyzed via Western blot. V5 tag antibody was used to probe biotinylated SLC25A13-APEX2-V5 protein levels, which remained constant under all three conditions: purine-rich, purine-deficient, and rapamycin-treated. This result was expected, as the self-biotinylating reaction of APEX2 conjugated to SLC25A13 occurs independently of cell growth conditions. TOMM20, serving as a mitochondrial outer membrane protein, also appeared to be independent of media. Both the V5 tag and TOMM20 were used as loading controls. However, the endogenous SLC25A13 protein level, probed using an SLC25A13 antibody, showed purine dependency, likely due to its higher expression compared to SLC25A13-APEX2-V5. Notably, the distribution of SLC25A13 transporters appeared to localize to a hotspot under purine-deficient conditions, resulting in a stronger band after purification. A similar trend was observed for the glycine transporter SLC25A38 (Fig. 4C). To further confirm this trend, we did a PLA assay between two transporters. Due to the antibody availability, we chose the SLC25A38 antibody paired with the V5 antibody representing SLC25A13 in the transduced HeLa cells. 62% ± 5% of cells were positive under purine-rich condition, while 87% ± 3% of cells became positive once the cells were grown under purine-deficient condition. Upon rapamycin treatment, 75% ± 6% of cells remained positive for the PLA pair of SLC25A13 and SLC25A38 (Fig. 4D) The results from two different methods presented a pattern that mitochondrial transporters were distributed through the inner membrane under purine-rich conditions, but SLC25A13 and SLC25A38 moved towards a common spot under purine-deficient conditions.

As a negative control, SLC25A19 was co-localized with SLC25A13 utilizing the V5 tag to perform PLA. Positive cells were 97% ± 3% under purine-deficient conditions and 96% ± 4% under purine-rich conditions. After SLC25A38 knockdown, the co-localization of SLC25A19 with SLC25A13 remained at 90% ± 9% under purine-deficient conditions (Fig. S3B). SLC25A19 is implicated in purine de novo synthesis, as previously discussed. Its co-localization with the aspartate transporter SLC25A13 remained consistent under both purine-rich and purine-deficient conditions. The knockdown of the glycine transporter also had no impact on the localization of SLC25A19. Consequently, the unrelated transporter SLC25A19 did not translocate to the mitochondrial hotspot under conditions that found purinosome function.

Furthermore, all six purinosome proteins were detected in the purified biotinylated protein samples. Among them, PPAT, GART, and ADSL showed purine dependency, while PAICS, ATIC, and PFAS did not exhibit purine dependency in this assay. This finding suggests a gathering of purinosome proteins towards mitochondria under purine-deficient conditions. However, the precise pattern of protein movement outside of mitochondria during purinosome formation remains under investigation. Hypoxanthine phosphoribosyltransferase (HPRT), a protein involved in the purine salvage pathway, was not recovered in the biotinylated protein purification. HPRT does not require substrates from mitochondria and is not part of the purinosome, explaining its absence in the purification (Fig. 4C).

Discussion

We have progressed the identity of the spatial relationship between the purinosome and mitochondria. Recall the purinosome is a liquid condensate consisting of six enzymes involved in the de novo purine biosynthesis, amidophosphoribosyltransferase (PPAT), trifunctional phosphoribosylglycinamide synthetase/formyltransferase/phosphoribosylaminoimidazole synthetase (GART), phosphoribosylformylglycinamidine synthase (PFAS), bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS), adenylosuccinate lyase (ADSL), and bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) (5, 35). Previous publications documented that purinosomes move along tubulin toward mitochondria and might directly uptake glycine from the mitochondrion instead of from the cytoplasm (6, 13). We confirmed our hypotheses that the purinosome might be located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, to facilitate the direct uptake of glycine, aspartate, and glutamate; necessary factors required by GART, PAICS, and ATIC in the de novo purine synthesis.

Given the dimensions of the mitochondrion and the <40 nm range of the PLA assay, the detected signal between the purinosome proteins and mitochondrial transporters would only be generated when purinosome proteins were close to mitochondria.

GART plays a vital role in the conversion of 5-phosphoribosylamine (PRA) to 5-phosphoribosylglycinamide (GAR) during the third step of the channeled purine de novo synthesis pathway. The reaction depends on the direct supply of glycine from mitochondria (6, 36). The essential proximity between GART and the glycine transporter SLC25A38 is dictated by the necessity for glycine supplementation under all cell growth conditions. In a purine-rich medium disfavoring purinosome assembly, 57% ± 7% of HeLa cells exhibited a positive GART-SLC25A13 PLA signal, yet 87% ± 17% of cells were PLA positive for the same pair of proteins under purinosome enhancing purine-deficient conditions, emphasizing the significance of their close association. Cells also displayed a positive co-localized PLA signal for the aspartate/glutamate transporter SLC25A13 with GART, suggesting that both transporters are in close proximity to GART. Rapamycin which disrupts the purinosome does not significantly dislodge GART from its locus on the membrane. Given the dimensions of the mitochondrion and the <40 nm range of the PLA assay, the detected signal between the purinosome proteins and mitochondrial transporters would only be generated when purinosome proteins were close to mitochondria.

There are also purinosome proteins that did not require direct supplementation from mitochondria, such as ADSL and ATIC, typically localized in the cytoplasm, that undergo relocation to the outside of mitochondria during purine-deficient conditions coinciding with the formation of purinosomes. Due to the limited availability of antibodies, we were unable to conduct a PLA assay to assess the proximity of ATIC to mitochondrial transporters. Consequently, we utilized ADSL localization as a representative marker in this study. ADSL was barely found in close proximity to both mitochondrial transporters, exhibiting under purine-rich conditions a 5% ± 5% PLA signal for ADSL-SLC25A38 and a 9% ± 10% PLA signal for ADSL-SLC25A13. In contrast, a noteworthy increase in both co-localization ratios was observed under purine-deficient conditions, with the PLA signals for ADSL-SLC25A38 and ADSL-SLC25A13 reaching 78% ± 8% and 58% ± 10%, respectively. consistent with purinosome formation. Treatment with rapamycin, which disrupts purinosome integrity (12, 28), resulted in decreased co-localization of ADSL with mitochondrial transporters. This result further confirmed that the proximity of ADSL to these transporters is sensitive to purinosome status. Notably, the PLA assay employed in our study does not necessitate protein overexpression thus providing a direct reflection of protein localization status at the endogenous level.

We chose APEX as a complementary proximity labeling method to PLA in this study. We designed an SLC25A13-conjugated APEX2 construct with a V5 tag (SLC25A13-APEX2-V5) to function as a probe for biotinylating purinosome proteins that were contiguous to the transporter. The APEX2 construct conjugated to the C-terminal of SLC25A13, was inserted into the intermembrane space of the mitochondria and led to biotinylation of all six purinosome proteins in the vicinity of the mitochondrial transporters under purine-deficient conditions, strongly supporting the presence of an intact purinosome.

The incorporation of the V5 tag also enabled us to discern the localization between the two transporters, SLC25A13 and SLC25A38. The aligned results from both APEX and PLA indicated transporter movement to a common spot on the mitochondrial inner membrane. That all six purinosome proteins were biotinylated is consistent with a contiguous condensate, spherical rather than an elongated assembly. That allows all six proteins to be in close proximity to the mitochondrial transporter SLC25A13-APEX2-V5 construct.

In summary, we propose a model wherein 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 (Fig. 4E). Considering that APEX reactions can be employed for non-targeted searches, we plan to continue our investigation using this transporter-APEX cell model to identify additional proteins that play significant roles in the purine de novo synthesis pathway. Although the APEX reaction necessitates transduction, the results of targeted analyses align with those obtained through PLA at the endogenous level. Therefore, we believe that the SLC25A13-APEX2-V5 construct transduced into HeLa cells can accurately reflect the endogenous cellular status without significant disturbance.

Experimental procedures

Reagents

HeLa and HEK293T cells were purchased from American Type Culture Collection (ATCC); DMEM with 4.5 g/L glucose, L-Gln, pyruvate, Corning Inc., catalog #10-013-CV; fetal bovine serium (FBS), R&D systems, catalog #S11550; trypsin-EDTA solution, phenol red, Corning Inc., caralog #25-053-CI; Spectra/Por Dialysis Tubing (MWCO 12-14 KD), Spectrum Chemicals & Laboratory Products, catalog #132676; sodium chloride, BDH Laboratory Supplies, catalog #BDH9286; Duolink in situ PLA probe anti-mouse MINUS, Sigma-Aldrich, Inc., catalog #DUO92004; Duolink in situ PLA probe anti-rabbit PLUS, Sigma-Aldrich, Inc., catalog #DUO92002; Duolink in situ wash buffer, Sigma-Aldrich, Inc., catalog #DUO82049; Duolink In Situ detection reagents TexasRed, Sigma-Aldrich, Inc., catalog #DUO92008; paraformaldehyde 16%, aq sol, Electron Microscopy Sciences, catalog #15710; Dulbecco’s Phosphate-Buffered Saline, 1× without calcium and magnesium (DPBS), Corning Inc., catalog #21-031-CV; Tris, Tris(hydroxymethyl)aminomethane, Sigma-Aldrich, Inc., catalog #T3253; Triton X-100, Sigma-Aldrich, Inc., catalog #X-100; DAPI dye 4,6-diamidino-2-phenylindole dihydrochloride, AdipoGen, catalog # CDXD0025M010; Quickchange lightning multisite-directed mutagenesis kit, Agilent Technologies, catalog #210515; T4 DNA ligase, New England Biolabs, catalog #M0202S; Gateway LR Clonase II, Thermo Scientific Pierce, catalog #11791-020; OptiMEM medium, Gibco Products, catalog #11058021; L-Glutamine, Gibco Products, catalog #25030081; hexadimethrine bromide (polybrene), Sigma-Aldrich, Inc., catalog #107689; puromycin dihydrochloride, Alfa Aesar, catalog #J61278MC; Sodium Dodecyl sulfate (SDS), VWR, catalog #0227; sodium deoxycholate, Sigma-Aldrich, Inc., catalog #D6750; 3-[(3-chloroamidopropyl)dimethylammonio)]-1-propanesulfonate (CHAPS), MP Biomedicals, Inc., catalog #ICN19031905; TCEP HCl Tris(2-carboxyethyl)phosphine hydrochloride, Gold Biotechnology Inc., catalog #TCEP-10; phenylmethylsulfonyl fluoride (PMSF), Amersco, catalog #M145; protease inhibitor, complete with EDTA, ROCHE Diagnostic, catalog # 04693124001; bovine serum albumin, Fisher Scientific, catalog #BP1600-100; polyoxyethylene sorbitan monolaurate (tween 20), Amresco, catalog #0777-1L; Biotin-phenol (Biotinyl tyramide), VWR, catalog #102987-498; hydrogen peroxide 30%, VWR, catalog #BOH7690-1; Trolox (6-Hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid), TCI America, catalog #H07265g; L-Ascorbic acid sodium salt, Alfa Aesar, catalog #A17759; Sodium azide, Sigma-Aldrich, Inc., catalog #S2002; Biotin antibody agarose, Immunechem, catalog #ICP0615; SLC25A38 siRNA cocktail, Santa Cruz Biotechnology catalog# sc-78287; Xfect RNA transfection reagent, TakaraBio catalog# 631450.

Antibodies used in the study: biotin antibody, Santa Cruz Biotechnology, catalog #SC101339; PFAS antibody, Bethyl Laboratories, Inc., catalog #A304-218A for Western blot, Novus Biologicals, Inc. catalog #NBP1-84691 for PLA; ADSL antibody, Bethyl Laboratories, Inc., catalog #A304-778A for Western blot, Novus Biologicals, Inc., catalog #NBP2-03107 for PLA; ATIC antibody, Bethyl Laboratories, Inc., catalog #A304-271A; PPAT antibody, GeneTex, catalog #GTX102725; GART antibody, Bethyl Laboratories, Inc., catalog #A304-311A; PAICS antibody, Bethyl Laboratories, Inc., catalog #A304-547A; V5 tag antibody, Cell Signaling, catalog #13202; HPRT antibody, Novus Biologicals, Inc., catalog #NBP2-46003; TOMM20 antibody, Santa Cruz Biotechnology, catalog #SC17764; SLC25A13 antibody, Novus Biologicals, Inc., catalog #NBP1-89019; SLC25A38 antibody, Novus Biologicals, Inc., catalog #NBP1-59559; beta-actin antibody, Fitzgerald Industries Intnl, catalog #10R-2927.

Cell culture

HeLa cells were cultured in DMEM medium supplemented with 10% FBS. Purine-deficient FBS was prepared by dialyzing FBS using a 10 kDa MWCO membrane in a 0.9% (w/v) NaCl solution at 4 °C. The NaCl solution was replaced every 12 h for a total of 4 times. Cells were seeded in DMEM with the supplement of 10% FBS or dialyzed FBS 24 h before conducting experiments.

Proximity ligation assay to detect endogenous protein co-localization

The proximity ligation assay was conducted following the manufacturer's suggested protocol. In brief, cells were seeded in either a purine-rich or purine-deficient medium (DMEM with 10% FBS or dialyzed FBS). Twenty-four hours post-seeding, cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 min. Subsequently, the fixed cells were rinsed once with PBS and twice with TBS (10 mM Tris pH 8, 150 mM NaCl). Permeabilization was carried out with 0.2% Triton X-100 in TBS at room temperature for 15 min, followed by washing with TBS. The cells were then blocked with PLA blocking solution at room temperature for 1 h. Primary antibodies, diluted in PLA diluent, were incubated with the cells at room temperature for 1 h. After removing the primary antibody solution, cells were washed twice with PLA wash buffer A at room temperature. Next, PLA ligation solution was prepared and incubated with cells at 37 °C in a humidified incubator for 30 min, followed by two washes with PLA wash buffer A at room temperature. Subsequently, PLA amplification solution was prepared and incubated with cells at 37 °C for 2 h. Following the incubation, cells were washed twice with PLA wash buffer B. The cell nuclei were stained with DAPI dye at room temperature for 5 min. Finally, the PLA sample slides were assembled and ready for imaging.

Proximity ligation assay sample imaging and analyzing

The PLA cell slides were imaged using a Nikon Eclipse TE-2000E inverted microscope with 60 × 1.49 Nikon Apo TIRF oil immersion objective with TexasRed (S535/30m) and DAPI (S500/20×) filter (Chroma Technology). For each frame of cells, a series of images were taken along the Z-axis for 13 nm thickness. Images were collected by an ORCA-flash 4.0LT digital camera (Hamamatsu, cat no: C11440) with Nikon NIS Elements (v. 4.5).

For analysis, the Z-stacked images were merged into one image using FIJI software (37). Based on the indication of DAPI, PLA puncta were counted for individual cells. A positive cell was counted if there were more than 6 PLA puncta inside. Total cell number was counted by DAPI staining. Eventually, the ratio of PLA-positive cells was reported as instructed by the manufacturer. On average, 30-50 cells were analyzed in each trial for one condition, and independent triplicates were performed for accuracy.

One-way ANOVA followed by Dunnett’s multiple comparisons test was performed using GraphPad Prism version 10.0.3 (217) for macOS, GraphPad Software, www.graphpad.com.

SLC25A38 knockdown using siRNA

SLC25A38 siRNA cocktail was purchased from Santa Cruz Biotechnology (catalog# sc-78287). Reverse transfection was performed to enhance the knockdown efficiency. 100pmol of siRNA was pre-incubated with Xfect RNA transfection reagent following the manufacturer instructions. (TakaraBio catalog# 631450) Mixed the siRNA/Xfect polymer with HeLa cells and seeded them in purine-rich or purine-deficient medium. 24h after seeding, cells were ready for Western blot or PLA assay.

Generating SLC25A13-APEX2-V5 plasmid for lentivirus transduction

pDONR221_SLC25A13 was a gift from RESOLUTE Consortium & Giulio Superti-Furga (Addgene plasmid # 132038; http://n2t.net/addgene:132038; RRID: Addgene_132038) (38). A V5 epitope (GKPIPNPLLGLDST) was inserted in-frame at the C-terminus of the SLC25A13 gene through site-directed mutagenesis using Phusion DNA polymerase. AgeI and XbaI restriction enzyme recognition sites were introduced between SLC25A13 and the V5 tag sequence using the same site-directed mutagenesis kit. Subsequently, the APEX2 encoding DNA construct was incorporated into the plasmid using AgeI and XbaI restriction enzymes, followed by T4 DNA ligation. The gene sequence was verified by full-length sequencing. Through a recombination reaction mediated by the LR clonase enzyme, the reconstruction of SLC25A13-APEX2-V5 was transferred from the entry vector to the destination lentiviral vector containing attR sites. The destination lentiviral vector was purified and ready for transduction.

Lentivirus transduced HeLa cell generation and selection

HEK293T cells were initially seeded in a T25 flask and then transfected with Lipofectamine 2000 according to the manufacturer’s instructions. Specifically, a mixture containing 10 μl of Lipofectamine 2000, 2 μg of the plasmid encoding SLC25A13-APEX2-V5, 1.5 μg of packaging plasmid psPAX2 (psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID:Addgene_12260)), and 0.5 μg of envelope plasmid pMD2.G (pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259; RRID:Addgene_12259)) was prepared in OptiMEM media and added drop-wise to the HEK293T cells. After a 1 min incubation with the transfection mixture, fresh DMEM media containing 10% FBS was added with 2 mM glutamine, and the cells were further incubated at 37 °C for 24 h. The old medium was removed and bleached immediately. Fresh DMEM/10% FBS with 2 mM glutamine was added to the cells and incubated with the cells for another 24h. During this time, the HEK293T cells packaged the SLC25A13-APEX2-V5 into viral particles, which were subsequently released into the medium.

The medium containing assembled viral particles was collected and filtered through a 0.45-micron filter. Next, 2 ml of the filtered viral particle-containing medium was added to HeLa cells in a 6-well plate along with 10 μg/ml polybrene. The HeLa cells were then incubated with the virus particles for 24 h. Subsequently, fresh DMEM medium containing 10% FBS was used to select transduced HeLa cells by adding 0.5 μg/ml puromycin for a week. The selection process was considered complete when non-transduced HeLa cells died completely. The waste generated in the entire procedure was autoclaved immediately each day.

Western blot to check SLC25A13-APEX2-V5 expression in HeLa cells

Wildtype HeLa cells and SLC25A13-APEX2-V5 transduced HeLa cells were separately seeded in DMEM medium supplemented with 10% FBS. After 24 h of seeding, cells were harvested and lysed in RIPA lysis buffer (50 mM Tris pH, 150 mM sodium chloride, 0,1% SDS, 0.5% sodium deoxycholate, 2% CHAPS, 10 mM TCEP, 1 mM PMSF, 1× protease inhibitor cocktail) on ice. The total protein concentrations were determined using the Bradford assay with BSA as the standard. Subsequently, 20 μg of total cell lysate was loaded onto a 12% SDS-PAGE gel for separation and then transferred onto a PVDF membrane for immunodetection. The PVDF membrane was blocked with 5% BSA in TBS after transfer. Next, the membrane was incubated overnight at 4 °C with either a 1:1000 dilution of SLC25A13 antibody or V5 tag antibody in 5% BSA/TBS, followed by washing with TBST buffer (10 mM Tris pH 8, 150 mM NaCl, 0.05% Tween 20). Finally, the membrane was probed with a 1:1000 dilution of horseradish peroxidase (HRP) conjugated secondary antibody for 1 h, washed with TBST buffer, and then ready for HRP signal development.

Immunofluorescent labeling of proteins in fixed cells

50,000 HeLa cells transduced with SLC25A13-APEX2-V5 were seeded onto 35 mm glass bottom dishes in DMEM medium supplemented with either 10% purine-rich or purine-deficient FBS. After 24 h of seeding, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min. Subsequently, the fixed cells were rinsed once with PBS followed by two washes with TBS. Permeabilization was achieved by incubating the cells with 0.2% Triton X-100 in TBS at room temperature for 15 min, followed by additional washing with TBS. The cells were then blocked with 1% bovine serum albumin (BSA) in TBS at room temperature for 30 min. Primary antibodies from different species were diluted in 1% BSA/TBS and incubated with the cells at room temperature for 1 h (1:500 anti-SLC25A13, rabbit; 1:500 anti-V5 tag, mouse; 1:500 anti-SLC25A38, rabbit; 1:500 anti-TOMM20, mouse). Following incubation, the cells were washed with TBS containing 0.1% Tween 20 (TBST) and then probed with fluorescent secondary antibodies at room temperature for 30 min (1:1000 anti-rabbit Alexa 488 or anti-mouse Alexa 647). DAPI staining was utilized for nuclear identification. Finally, the cells were washed twice with TBST and once with TBS before being mounted for imaging.

Fixed cell imaging employing three-color fluorescence was performed using a Nikon Eclipse TE2000 inverted microscope equipped with a VisiTech Instantaneous Selective Illumination Microscopic system (VT-iSIM) and an ORCA-FLas 4.0 VT Digital CMOS camera (C11440-22CU, Hamamatsu). Imaging was conducted with a 100× CFI Apochromat TIRF 1.49 Oil immersion objective (Nikon), providing a theoretical lateral resolution of 125 nm. Excitation of the cell samples was carried out at wavelengths of 405 nm, 488 nm, and 642 nm for 500 ms.

Proximity labeling assay (APEX) to label protein near SLC25A13 vicinity

HeLa cells transduced with SLC25A13-APEX2-V5 were cultured in DMEM medium supplemented with 10% purine-rich or purine-deficient FBS. After 24 h of seeding, the APEX reaction was initiated by adding 1 mM biotin-phenol to the growth medium. The cells were then incubated with biotin-phenol at 37 °C for 2 h, followed by the addition of 1 mM H2O2 to initiate the biotinylation process. To quench the reaction, an equal volume of PBS containing 5 mM Trolox, 10 mM sodium ascorbate, and 10 mM sodium azide was added to the medium, and the cells were incubated with the complete quenching medium at room temperature for 5 min before rinsing with PBS. Subsequently, the cells were fixed and subjected to immunofluorescent labeling using the same protocol as described above. The primary antibody used was 1:500 biotin antibody. Imaging was conducted using the same VT-iSIM system as above.

Biotinylated protein purification and western blotting

Following the APEX reaction as described earlier, the cells were quenched and rinsed with PBS before being harvested via trypsinization and lysed in RIPA buffer (50 mM Tris, 150 mM sodium chloride, 0.1% SDS, 0.5% sodium deoxycholate, 2% CHAPS, 10 mM TCEP, 1 mM PMSF, 1× protease inhibitor cocktail, 5 mM Trolox, 10 mM sodium ascorbate, 10 mM sodium azide). The cells were incubated with RIPA buffer on ice for 20 min with 5 min of sonication. Subsequently, the cell lysate was cleared by centrifugation at 4 °C, 13,000 rpm for 20 min, and the total protein concentration was determined using the Bradford assay with BSA as a standard. For Western blot analysis, 20 μg of total cell lysate was used and probed with the biotin antibody following the same protocol as described previously.

The remaining cell lysate was subjected to biotinylated protein purification. Biotin antibody-conjugated agarose beads were pre-blocked with 5% BSA/TBS supplemented with 1× protease inhibitor cocktail and 1 mM PMSF at 4 °C overnight before purification. After blocking, the agarose beads were centrifuged, and the blocking solution was discarded. The beads were then equilibrated three times in the RIPA lysis buffer. Subsequently, the cell lysate was loaded onto the beads and incubated at 4 °C overnight. The biotinylated protein-bound beads were harvested by centrifugation and washed 10 times in TBS containing 1× protease inhibitor, 1 mM PMSF, 10 mM TCEP, and 0.5% CHAPS. Finally, the biotinylated proteins were eluted from the beads by incubating with 8 mg/ml biotin, 1× protease inhibitor, 1 mM PMSF, 10 mM TCEP, and 0.5% CHAPS at room temperature for 3 h. The total protein concentration in the elution was determined using the Bradford assay. For Western blot analysis, 10 μg of elution proteins were probed with purinosome protein antibodies, mitochondrial transporter antibodies, and selected control protein antibodies. The antibody dilution ratio was 1:1000 for all.

Data availability

The data discussed in this study are available in the article. Additional data files are available upon request.

Supporting information

This article contains supporting information.

Conflict of interests

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Supporting Figures

Acknowledgments

We acknowledge the helpful discussion with Vidhi Pareek, Phil Hanoian, and Syed Ali Naqi Jaffery. We also thank Anthony Pedley for the packaging plasmid (psPAX2 Addgene# 12260), and envelope plasmid (pMD2.G Addgene# 12259); and Missy Hazen, as well as Penn State Microscopy Facility at University Park, PA, for technical expertise.

Author contributions

S. J. B. and Z. S. conceptualization, S. J. B. funding acquisition, S. J. B. investigation, S. J. B. and Z. S. methodology, S. J. B. project administration, S. J. B. resources, S. J. B. supervision, S. J. B. writing–review & editing. Z. S. data curation; Z. S. formal analysis; Z. S. validation; Z. S. visualization; Z. S. writing–original draft.

Funding and additional information

This work was supported by 10.13039/100000007 National Institutes of Health Grant R01GM024129 (S. J. B.).
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