Protein Aggregation and Polyasparagine-Mediated Cellular Toxicity inSaccharomyces cerevisiae

Abstract

It is well established that protein aggregation is associated with many neurodegenerative disorders including polyglutamine diseases, but a mechanistic understanding of the role of protein aggregates in the disease pathogenesis remains elusive. Previously thought to be the cause of cellular toxicity such as cellular dysfunction and cell death, protein aggregation is now proposed to serve a protective role by sequestering toxic oligomers from interfering with essential physiological processes. To investigate the relationship between protein aggregation and cellular toxicity, we have characterized and compared the effects of two GFP-fusion proteins that form aggregates in Saccharomyces cerevisiae, one with a polyasparagine repeat (GFP N104 ) and one without (GFP C ). Although both proteins can form microscopically visible GFP-positive aggregates, only the GFP N104 -containing aggregates exhibit morphological and biochemical characteristics that resemble the aggregates formed by mutant huntingtin in yeast cells. Formation of both the GFP C and GFP N104 aggregates depends on microtubules, while only the GFP N104 aggregate requires the chaperone Hsp104 and the prion Rnq1 and is resistant to SDS. Although no microscopically visible GFP N104 aggregates were observed in the hsp104D and rnq1D mutant cells, SDS-insoluble aggregates can still be detected by the filter trap assay. These observations argue that the GFP N104 -containing aggregates can exist in at least two distinct states in vivo. We also show that a nucleus-targeted GFP N104 interferes with transcription from two SAGA-dependant promoters and results in a decrease in cell viability. Overall, the results imply that the GFP N104 protein behaves similarly to the mutant huntingtin in yeast cells and provides a new model for investigating the interplay between protein aggregates and the associated phenotypes.

Compositional Determinants of Prion Formation in Yeast

“…Stacking of Q/N residues to form polar zippers has been proposed to stabilize amyloid fibrils (35). Consistent with this hypothesis, mutational studies of Sup35 indicate that Q/N residues are critical for driving [PSI ϩ ] formation (12), and expanded poly-Q or poly-N tracts are sufficient to drive amyloid aggregation (36,63). Therefore, this paper examines the se-quence features that allow the polar, Q/N-rich yeast PFDs to form prions.…”

The Importance of TM3-4 Loop Subdomains for Functional Reconstitution of Glycine Receptors by Independent Domains

“…Our data clearly show that the variable region V2 and the conserved region C2 were not responsible for this effect. (29,30). Lack of PPPAP as in the construct iD⌬49-TM4 decreased GlyR rescue efficiency to 58%, suggesting that this motif may be of some importance for the correct GlyR configuration, yet the lower efficiency of ion channel rescue could also be a consequence of continuous protein truncation and therefore loss of steric contact surfaces.…”

A Small, Glutamine-Free Domain Propagates the [SWI+] Prion in Budding Yeast

“…Our results correlate well with this observation. While one study did show that a polyasparagine peptide [poly(N104)] could form SDS-insoluble, Hsp104-dependent aggregates in yeast, this peptide does not demonstrate the heritability that is characteristic of prions and, at 104 amino acids, was much larger than the asparagine-rich PrD described here (35).…”