Limits...
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.

Show MeSH

Related in: MedlinePlus

Molecular model of [URE3] amyloid filament composed of Ure2p. Polymerized prion domains (red) form the protease-resistant core of the filament and are inaccessible to antibodies because they are masked by the nitrogen regulation domain (blue), and perhaps also because of their compact structure. β Strands in the prion domain are perpendicular to the long axis of the filament. The nitrogen regulation domain is exposed both to antibody binding and to protease digestion.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192028&req=5

Figure 5: Molecular model of [URE3] amyloid filament composed of Ure2p. Polymerized prion domains (red) form the protease-resistant core of the filament and are inaccessible to antibodies because they are masked by the nitrogen regulation domain (blue), and perhaps also because of their compact structure. β Strands in the prion domain are perpendicular to the long axis of the filament. The nitrogen regulation domain is exposed both to antibody binding and to protease digestion.

Mentions: The above observations support the proposal (Taylor et al. 1999) whereby the NH2-terminal domains stack axially to form the filament backbone from which the COOH-terminal domains protrude (Fig. 5). Sequestration of the NH2 termini in this way explains their inaccessibility to the NH2-terminal antibody. It also accounts for the lower reactivity of the NH2-terminal antibody with cryo-sections, although as expected, some labeling was observed as cross-cutting should expose some NH2-terminal epitopes.


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

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

Molecular model of [URE3] amyloid filament composed of Ure2p. Polymerized prion domains (red) form the protease-resistant core of the filament and are inaccessible to antibodies because they are masked by the nitrogen regulation domain (blue), and perhaps also because of their compact structure. β Strands in the prion domain are perpendicular to the long axis of the filament. The nitrogen regulation domain is exposed both to antibody binding and to protease digestion.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Molecular model of [URE3] amyloid filament composed of Ure2p. Polymerized prion domains (red) form the protease-resistant core of the filament and are inaccessible to antibodies because they are masked by the nitrogen regulation domain (blue), and perhaps also because of their compact structure. β Strands in the prion domain are perpendicular to the long axis of the filament. The nitrogen regulation domain is exposed both to antibody binding and to protease digestion.
Mentions: The above observations support the proposal (Taylor et al. 1999) whereby the NH2-terminal domains stack axially to form the filament backbone from which the COOH-terminal domains protrude (Fig. 5). Sequestration of the NH2 termini in this way explains their inaccessibility to the NH2-terminal antibody. It also accounts for the lower reactivity of the NH2-terminal antibody with cryo-sections, although as expected, some labeling was observed as cross-cutting should expose some NH2-terminal epitopes.

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