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Prion filament networks in [URE3] cells of Saccharomyces cerevisiae.

Speransky VV, Taylor KL, Edskes HK, Wickner RB, Steven AC - J. Cell Biol. (2001)

Bottom Line: In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates.However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments.This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH(2)-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

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Immunoelectron microscopy of control [ure-o] cells. Most labeled cryosections (>80%) exhibit a sparse distribution of gold particles all over the cell (e.g., A). Approximately 5% of sections show concentrations of label at the cell periphery (B). Another 5–10% show one or more focal clusters within which the gold particles appear to be distributed randomly (C), in addition to diffuse labeling elsewhere. Bar, 0.5 μm.
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Figure 3: Immunoelectron microscopy of control [ure-o] cells. Most labeled cryosections (>80%) exhibit a sparse distribution of gold particles all over the cell (e.g., A). Approximately 5% of sections show concentrations of label at the cell periphery (B). Another 5–10% show one or more focal clusters within which the gold particles appear to be distributed randomly (C), in addition to diffuse labeling elsewhere. Bar, 0.5 μm.

Mentions: To quantitate the incidence of aggregates in [URE3] cells, micrographs of 127 randomly chosen cell sections were recorded at a magnification of 12,000. 54 of these were eliminated as either too small (tangential sections) or poorly preserved. Of the remaining 73, 4 had aggregates of 3–6 μm in diameter, 3 had aggregates of 0.8–1.5 μm, 3 had aggregates of 0.4–0.8 μm, and 6 had short lines of 3–5 closely spaced gold particles. In comparison, no such alignments were seen in >300 examples of the majority kind of control [ure-o] sections (see Fig. 3 A), whereas the two other kinds of [ure-o] labeling patterns (see Fig. 3B and Fig. C) were qualitatively different, rare, and readily recognizable. To assess the average densities of dispersed gold particles, we measured the areas and counted the gold particles of 20 [URE3] sections (minus areas with aggregates) and 15 [ure-o] sections.


Prion filament networks in [URE3] cells of Saccharomyces cerevisiae.

Speransky VV, Taylor KL, Edskes HK, Wickner RB, Steven AC - J. Cell Biol. (2001)

Immunoelectron microscopy of control [ure-o] cells. Most labeled cryosections (>80%) exhibit a sparse distribution of gold particles all over the cell (e.g., A). Approximately 5% of sections show concentrations of label at the cell periphery (B). Another 5–10% show one or more focal clusters within which the gold particles appear to be distributed randomly (C), in addition to diffuse labeling elsewhere. Bar, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192028&req=5

Figure 3: Immunoelectron microscopy of control [ure-o] cells. Most labeled cryosections (>80%) exhibit a sparse distribution of gold particles all over the cell (e.g., A). Approximately 5% of sections show concentrations of label at the cell periphery (B). Another 5–10% show one or more focal clusters within which the gold particles appear to be distributed randomly (C), in addition to diffuse labeling elsewhere. Bar, 0.5 μm.
Mentions: To quantitate the incidence of aggregates in [URE3] cells, micrographs of 127 randomly chosen cell sections were recorded at a magnification of 12,000. 54 of these were eliminated as either too small (tangential sections) or poorly preserved. Of the remaining 73, 4 had aggregates of 3–6 μm in diameter, 3 had aggregates of 0.8–1.5 μm, 3 had aggregates of 0.4–0.8 μm, and 6 had short lines of 3–5 closely spaced gold particles. In comparison, no such alignments were seen in >300 examples of the majority kind of control [ure-o] sections (see Fig. 3 A), whereas the two other kinds of [ure-o] labeling patterns (see Fig. 3B and Fig. C) were qualitatively different, rare, and readily recognizable. To assess the average densities of dispersed gold particles, we measured the areas and counted the gold particles of 20 [URE3] sections (minus areas with aggregates) and 15 [ure-o] sections.

Bottom Line: In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates.However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments.This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH(2)-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

Show MeSH
Related in: MedlinePlus