Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies

Zhao, Alice; Tsechansky, Mark; Swaminathan, Jagannath; Cook, L S; Ellington, Andrew D.; Marcotte, Edward M.

doi:10.1371/journal.pone.0056203

Cited by 11 publications

(35 citation statements)

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“…Follow-up studies have since suggested that the reported foci formation of transiently expressed human purine biosynthesis enzymes was independent of purines and thus may explain this discrepancy [22]. In order to more carefully assay for a potential purinosome, we additionally tested for the co-localization of yeast purine biosynthesis enzymes in foci.…”

Section: Resultsmentioning

confidence: 99%

A proteomic survey of widespread protein aggregation in yeast

et al. 2014

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Many normally cytosolic yeast proteins form insoluble intracellular bodies in response to nutrient depletion, suggesting the potential for widespread protein aggregation in stressed cells. Nearly 200 such bodies have been found in yeast by screening libraries of fluorescently tagged proteins. In order to more broadly characterize the formation of these bodies in response to stress, we employed a proteome-wide shotgun mass spectrometry assay in order to measure shifts in the intracellular solubilities of endogenous proteins following heat stress. As quantified by mass spectrometry, heat stress tended to shift the same proteins into insoluble form as did nutrient depletion; many of these proteins were also known to form foci in response to arsenic stress. Affinity purification of several foci-forming proteins showed enrichment for co-purifying chaperones, including Hsp90 chaperones. Tests of induction conditions and co-localization of metabolic enzymes participating in the same metabolic pathways suggested those foci did not correspond to multi-enzyme organizing centers. Thus, in yeast, the formation of stress bodies appears common across diverse, normally diffuse cytoplasmic proteins and is induced by multiple types of cell stress, including thermal, chemical, and nutrient stress.

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Section: Resultsmentioning

confidence: 99%

A proteomic survey of widespread protein aggregation in yeast

et al. 2014

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“…In contrast, previous efforts using cells stably transfected with purinosome constructs did not yield visible purinosome bodies (Gooljarsingh et al 2001). Overexpression of proteins above their native levels has been shown to result in their aggregation (Cromwell et al 2006, Klein & Dhurjati 1995, Mayer & Buchner 2004, Yokota et al 2000, Zhang et al 2004), and the transiently overexpressed purine biosynthetic enzymes form intracellular bodies to extents that correspond to each enzyme’s predicted aggregation propensity (Zhao et al 2012). The resultant bodies are marked by ubiquitin and heat-shock chaperones, which suggests that they may represent aggregated protein clusters (Zhao et al 2012).…”

Section: Fibers and Focimentioning

confidence: 99%

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

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Zhao

Ellington

et al. 2012

Annu. Rev. Cell Dev. Biol.

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Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.

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“…For example, the six enzymes of the human de novo purine biosynthetic pathway were observed to reversibly form clusters, named purinosomes, in HeLa cells in response to purine availability 17,21 , though the role of expression levels and fluorescent fusions remains open 22 . Furthermore, in a recent study 18 of Saccharomyces cerevisiae it was observed that out of 800 GFP-tagged cytosolic proteins, 180 clearly displayed evidence of dynamic clustering, with many of these proteins involved in intermediary metabolism and stress response.…”

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Enzyme clustering accelerates processing of intermediates through metabolic channeling

et al. 2014

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We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.

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Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies

Cited by 11 publications

References 20 publications

A proteomic survey of widespread protein aggregation in yeast

A proteomic survey of widespread protein aggregation in yeast

Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

Enzyme clustering accelerates processing of intermediates through metabolic channeling

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