Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90

Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid-liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low-complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Finally, we demonstrate that the heat shock protein 90 KDa helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of heat shock protein 90 KDa activity induced the transformation of formylglycinamidine ribonucleotide synthase clusters into more irregularly shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells.

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Work Title Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90
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Open Access
Creators
  1. Anthony Pedley
Keyword
  1. liquid condensate; liquid–liquid phase separation; metabolism; protein complex; purine biosynthesis
License CC BY-NC-ND 4.0 (Attribution-NonCommercial-NoDerivatives)
Work Type Article
Acknowledgments
  1. Huck Institutes of the Life Sciences at Penn State (Huck Innovative and Transformative Seed Fund grant to AMP)
  2. The National Institutes of Health (grant nos.: R01GM024129 [to S. J. B.], R01GM134086 [to MK], T32GM066706 [to ELK], and R25GM55036 [to ELK])
Publisher
  1. J Biol Chem
Publication Date 2022
Publisher Identifier (DOI)
  1. 10.1016/j.jbc.2022.101845
Related URLs
Deposited November 06, 2022

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Version 1
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  • Created
  • Updated
  • Updated Acknowledgments Show Changes
    Acknowledgments
    • Huck Institutes of the Life Sciences at Penn State (Huck Innovative and Transformative Seed Fund grant to AMP), The National Institutes of Health (grant nos.: R01GM024129 [to S. J. B.], R01GM134086 [to MK], T32GM066706 [to ELK], and R25GM55036 [to ELK])
  • Added Creator Anthony Pedley
  • Added Fig 3_HSP90 Inhibition_v3.tif
  • Added Fig1_Condensation.tif
  • Added Fig2_Properties.png
  • Added Fig3_HSP90 Inhibition_v2.tif
  • Added Fig4_Model.png
  • Added FigS7_pH.png
  • Added Fig S1_HeTOFLI Expression.png
  • Added FigS2_Sphericity.png
  • Added Movie 1. Merging of Purinosome Condensates.avi
  • Added Fig S3_Merging.tif
  • Added Fig S4_Intrinsic Disorder-combined_v1.tif
  • Added Fig S6_v2.tif
  • Added Fig S8_trypan blue viability_STA9090_depleted HeLa.png
  • Added Fig S9_HSP90 Inhibition in P- HeLa.png
  • Added Fig S5_Aggregation Tendency Predictions_v1.tif
  • Deleted Fig3_HSP90 Inhibition_v2.tif
  • Updated License Show Changes
    License
    • https://creativecommons.org/licenses/by-nc-nd/4.0/
  • Published