Quantitative Effects of Magnesium Chloride Stress on Aggregation of Sup35p in [psi-] Yeast Cells

Yao      Song; Wanjun      Lan; Xianyuan      Wu; Jianwei      He; Hui      Li; Songbin      Ben; Youtao      Song

Abstract

[PSI+] phenotype can be transiently induced when Magnesium chloride (MgCl2) was the selective pressure in SUP35 repeat-expansion mutant [psi-] yeast strains. We further investigated [PSI+] phenotype change under different MgCl2 conditions with native Sup35p and quantified the Sup35p status changes with fluorescence recovery after photobleaching (FRAP) and semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) analysis. It was found that the [PSI+] phenotype presented a dose-dependent relationship with the concentrations of MgCl2. Furthermore, Sup35p aggregated in MgCl2 treated cells but did not form large aggregates as it does in [PSI+] cells, and the size of Sup35p aggregates showed a time-dependent relationship with the MgCl2 application. The aggregation of Sup35p strictly depended on the presence of MgCl2 stress in our strains.

Keywords: Magnesium chloride, prion, [PSI+], quantitative effects, Sup35p, stress, Sup35p in [psi-] Yeast Cells, fluorescence recovery after photobleaching, semi-denaturing detergent-agarose gel electrophoresis, PSI+, Saccharomyces cerevisiae, nonsense suppression, Immunoblotting, SDS-PAGE, SDD-AGE, Green Fluorescent Protein, guanidine curing, adenine, Confocal Microscopy, FRAP assay, Western Blot Analysis, Tris-borate-EDT, Immo-bilon polyvinylidene difluoride sheets, guanidine-hydrochloride, Live-Time Fluorescence Analysis, isogenic [Gpsi, –, ] cells, SDS-insoluble Sup35p, NGMC diffusion, SDS-resistant Sup35p polymers, anti-Sup35p antiserum