title: Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.
abstract: The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.
authors.affiliation: a Department of Biological Chemistry; Institute of Life Sciences ; Hebrew University of Jerusalem ; Jerusalem , Israel.
authors.forename: Ayala
authors.initials: A
authors.lastname: Shiber
authors.forename: William
authors.initials: W
authors.lastname: Breuer
authors.forename: Tommer
authors.initials: T
authors.lastname: Ravid
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry; D006360:Heat-Shock Proteins; D066329:Protein Aggregates; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D066329:Protein Aggregates; D049452:Green Fluorescent Proteins
keywords: FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; Hsp, heat shock protein; chaperones; flow cytometry; fluorescent tags; protein aggregation; protein degradation; protein misfolding; protein quality control