Abstract
Polyglutamine disease is now recognized as one of the conformational, amyloid-related diseases. In this disease, polyglutamine expansion in proteins has toxic effects on cells and also results in the formation of aggregates. Polyglutamine aggregate formation is accompanied by conversion of the polyglutamine from a soluble to an insoluble form. In yeast, the efficiency of the aggregate formation is determined by the balance of various parameters, including the length of the polyglutamine tract, the function of Hsp104, and the level of polyglutamine expression. In this study, we found that the co-expression of a long polyglutamine tract, which formed aggregates independently of the function of Hsp104, enhanced the formation of aggregates of a short polyglutamine tract in wild-type cells as well as in Deltahsp104 mutant cells. Thus, the expression of a long polyglutamine tract would be an additional parameter determining the efficiency of aggregate formation of a short polyglutamine tract. The co-localization of aggregates of long and short polyglutamine tracts suggests the possibility that the enhancement occurs due to the seeding of aggregates of the long polyglutamine tracts.
Highlights
Expansion of polyglutamine repeats in a protein has been proved to account for the pathogenesis of at least nine inherited neurodegenerative diseases, including spinobulbar muscular atrophy, Huntington’s disease, spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 (SCA1, SCA2, SCA3/MJD, SCA6, SCA7, and SCA17, respectively), and dentatorubural pallidoluysian atrophy [1,2,3,4]
Yeast—First, we examined whether polyglutamine aggregates in yeast were stained with thioflavin-S, a compound that binds to amyloid fibrils [22]
Aggregate formation of polyglutaminecontaining proteins is seeded and recruited by preformed polyglutamine fibrils, suggesting that the aggregates form by a process of nucleation-dependent polymerization [14, 15]
Summary
Yeast Strains, Media, and Growth—The wild-type strain W303 To create high copy plasmids (pTV3HA-Q34, pTV3HA-Q80), the SalI-EcoRI fragment of GPD-3HA-Q34 or GPD-3HA-Q80 was ligated with SalI-EcoRI-cleaved pTV3. P50-Q24 encoded by low and high copy plasmids, Gal-p50-Q24 and MGal-p50-Q24, respectively, were obtained as follows. The KpnI-NheI fragment of p50-U was ligated with the KpnI-SalI fragment and the vector portion of the NheI-SalI fragment from p50-Q80 to create Gal-p50-Q24. Cells were incubated with anti-p50 antibody for 1 h and washed with G1T three times. Cells were incubated with Texas Red X-conjugated anti-mouse IgG (Molecular Probes) for 30 min, and washed with G1T three times. For double-labeled immunofluorescence, cells were incubated first with mouse anti-p50 monoclonal antibody and rabbit anti-HA antibody (Medical and Biological Laboratories, Nagoya, Japan), and with Texas Red X-conjugated goat antimouse IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Vector Laboratories) as secondary antibodies.
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