Difference between revisions of "Widespreadaggregation.2013"

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== Abstract ==
 
== Abstract ==
Nearly 200 subcellular protein bodies have been found in yeast by screening libraries of fluorescently tagged proteins, but cellular roles for these bodies—typically proteins accumulated into large intracellular foci—remain mysterious. We systematically tested foci for roles as metabolic pathway organizing centers, as aggregates, or as protein storage bodies. Tests of induction conditions and co-localization suggested most foci probably do not represent multi-enzyme organizing centers; more likely, they represent aggregates or storage bodies. Heat shock or arsenic stress tended to shift the same proteins into insoluble form, as quantified by mass spectrometry of native proteins. Moreover, affinity purification of several foci-forming proteins showed enrichment for co-purifying chaperones. Finally, the occurrence of glutamine synthetase foci correlated with markers of cell age, while treatment with rapamycin antagonized their formation. Thus, in yeast, we observed broad rejection of the hypothesis that subcellular protein foci generally serve as functional organizing centers for metabolic pathways; rather, widespread aggregation appears common across diverse, normally diffuse cytoplasmic proteins and is increased with age and induced by multiple types of cell stress.
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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.
  
 
== Shotgun proteomics experimental design ==
 
== Shotgun proteomics experimental design ==

Revision as of 10:27, 3 February 2014

This is supplementary website for the following paper:

Jeremy O’Connell, Mark Tsechansky, Ariel Royal, Dan Boutz, Andrew D. Ellington, Edward M. Marcotte, A proteomic survey of widespread protein aggregation in yeast, Molecular BioSystems, in press (2014).


Contents

Abstract

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.

Shotgun proteomics experimental design

To be added

Shotgun proteomics datasets

Database search results

Summary

To be added

Contact