title: Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.
abstract: Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland Baltimore County , 1000 Hilltop Circle, Baltimore, Maryland 21250, United States.
authors.forename: Minjoung
authors.initials: M
authors.lastname: Kyoung
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Russell
authors.forename: Casey L
authors.initials: CL
authors.lastname: Kohnhorst
authors.forename: Nopondo N
authors.initials: NN
authors.lastname: Esemoto
authors.forename: Songon
authors.initials: S
authors.lastname: An
mesh_terms: D055592:Biophysical Phenomena; D053898:Biosynthetic Pathways; D045744:Cell Line, Tumor; D002470:Cell Survival; D003594:Cytoplasmic Granules / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D011687:Purines / Q000096:biosynthesis*
chemical_list: D009097:Multienzyme Complexes; D011687:Purines; D049452:Green Fluorescent Proteins; C030985:purine