Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity

Prion properties of the Sup35 protein of yeast Pichia methanolica

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C–terminal domain responsible for translation termination and N–terminal extensions of different structure. The N–terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N–terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], ‘hybrid’ [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a ‘species barrier’ for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family

PolyQ toxicity is modulated by the levels of Sup45.

<p>Growth of the [<i>psi</i>⁻] [<i>PIN</i>⁺] and [<i>psi</i>⁻] [<i>pin</i>⁻] transformants of the 74-D694 strain carrying either a centromeric plasmid with <i>SUP45</i> or an empty vector as a control and expressing either 103Q-GFP or 25Q-GFP, was analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#pone-0029832-g004" target="_blank">Fig. 4</a>.</p

PolyQ toxicity depends on [<i>PIN</i>⁺] and Sup35.

<p>The [<i>psi</i>⁻] [<i>PIN</i>⁺] and [<i>psi</i>⁻] [<i>pin</i>⁻] transformants of the 74-D694 strain (<i>SUP35</i>) or its 74-D694 ΔS35 derivative disrupted for chromosomal <i>SUP35</i> and carrying <i>SUP35-C</i> on a centromeric plasmid (<i>SUP35-C</i>), both expressing either 103Q-GFP or 25Q-GFP, were grown at 30°C in liquid SC-Ura medium with glucose resuspended in the same medium but with raffinose instead of glucose and after 12 h incubation, cell suspensions were diluted to an OD₆₀₀ of 1.0, and then spotted onto SC-Ura plates with galactose as a sole carbon source (Gal) and incubated for 4 days. Equal spotting was controlled by spotting the cells onto SC-Ura plates containing glucose as carbon source (Glc) in parallel. Five serial 5-fold dilutions of cell suspensions are shown.</p

Semi-quantitative dot-blot analysis of 103Q-GFP and 25Q-GFP.

<p>Transformants of 74-D694 [<i>psi</i>⁻] [<i>PIN</i>⁺] or [<i>psi</i>⁻] [<i>pin</i>⁻] with the multicopy p103Q-GFP or p25Q-GFP plasmids were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#s4" target="_blank">Materials and Methods</a> with incubation in SC-Ura Gal medium for indicated time. Equal amounts of total protein (confirmed by staining the same membranes by Ponceau S, a non-specific protein stain) from each lysate were serially diluted in four-fold increments and applied to a nitrocellulose membrane. Blots were probed with the monoclonal anti-GFP antibody.</p

Centrifugation analysis of aggregation of Sup35 and Sup45 in the presence of 103Q-GFP or 25Q-GFP.

<p>Transformants of the strain 74-D694 [<i>psi</i>⁻] [<i>PIN</i>⁺] with plasmids expressing 103Q-GFP or 25Q-GFP were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#s4" target="_blank">Materials and Methods</a>. After incubation in SC-Ura Gal medium for 9 h, cells were harvested. Cell lysates were fractionated by centrifugation and fractions were analyzed by Western blotting: staining with either anti-Sup35NM (Sup35) or anti-Sup45 (Sup45) polyclonal antibodies. The lower bands in the Sup35 image represent a degradation product (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#pone-0029832-g003" target="_blank">Fig. 3</a>); the lower bands in the Sup45 image are nonspecific protein staining <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#pone.0029832-Valouev1" target="_blank">[20]</a>. T, total protein; S, soluble fraction (1∶2 and 1∶4, dilutions); P, pellet. CEN-SUP45, the centromeric plasmid pRS315-SUP45; empty vector, pRS315. Quantitative data on Sup45 levels obtained by densitometric analysis demonstrated that expression of Q103-GFP caused an approximately 2-fold depletion of the soluble fraction of Sup45, with the remaining portion of the protein being relocated into the pellet fraction. Presence of pRS315-SUP45 caused an approximately 4-fold increase in the level of soluble Sup45 in Q103-GFP expressing cells as compared with the same cells with an empty vector.</p

103Q-GFP-dependent polymerization of Sup35.

<p>(<b>A</b>) Polymers of Sup35 visualized by SDD-AGE. 74-D694 [<i>psi</i>⁻] [<i>PIN</i>⁺] and [<i>psi</i>⁻] [<i>pin</i>⁻] transformants with the p103Q-GFP plasmid were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#s4" target="_blank">Materials and Methods</a>. After incubation in SC-Ura Gal medium for 9 h, cells were harvested and their lysates were used to analyze the amount of SDS-insoluble polymers of Sup35. (<b>B</b>) The levels of Sup35 monomer, SDS-PAGE analysis. Lysates were obtained from cells grown as described above. The samples were not boiled before loading onto the gel which only allowed SDS-soluble Sup35 to enter the gel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#pone.0029832-Kushnirov1" target="_blank">[34]</a>. Lower bands represent a Sup35 degradation product, characteristic of the non-prion/amyloid form of Sup35 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029832#pone.0029832-Paushkin1" target="_blank">[36]</a>. Total lysates (T) and their serial dilutions are indicated. 74-D694 [<i>psi</i>⁻] [<i>pin</i>⁻] expressing 25Q-GFP and 74-D694 [<i>PSI</i>⁺], respectively, are shown for comparison. Blots were stained with anti-Sup35NM antibody. Quantification of Sup35 by densitometric analysis demonstrated that upon expression of 103Q-GFP the levels of soluble Sup35 were approximately 3-fold lower in [<i>PIN</i>⁺] than in [<i>pin</i>⁻] cells.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p