Fig 1.
The rpt6-S119A mutants had increased susceptibility to proteotoxic stress and displayed abnormal morphology.
(A) Alignment of Rpt6 sequences from rat, human and yeast indicate conservation of the S120 phosphorylation site. Note the presence of a serine at position 117 in yeast. This residue may contribute to some cross-immunoreactivity for the pS120 antibody in the myc-rpt6-S119A strain (see below). (B) Qualitative analysis of myc-tagged Rpt6 (myc-Rpt6) expression. Immunoblot depicts similar levels of expression in strains tested. (C) Representative immunoblot of myc-RPT6 and myc-rpt6-S119A lysates immunoprecipitated with α-myc antibodies and resolved on SDS-PAGE. The anti-Rpt6 pS120 antibody recognizes wild-type myc-Rpt6, whereas little to no signal is observed for the myc-rpt6-S119A mutant, suggesting that Rpt6 is phosphorylated in yeast. (D) Increased sensitivity to proteotoxic stress is observed in the myc-rpt6-S119A mutant. The fitness of the myc-rpt6-S119D strain was comparable to myc-RPT6. The myc-rpt6-S119A strain was sensitive to elevated temperature, ethanol and canavanine. Arg- medium serves as a control for canavanine, which is a toxic analog of arginine. (E) Phosphorylation of endogenous Rpt6 is increased upon stress. Protein lysates from cells grown under normal and stress conditions (37° and canavanine (1ug/ml;2h)) were probed with the anti-pS120 antibody. (F) Representative micrographs of myc-RPT6 strains (DIC and DAPI) during log phase indicate abnormal morphology in the myc-rpt6-S119A mutant. (G) Quantitative analysis of morphological studies in (F). Graphs denote 3 independent experiments. Abnormal morphology is increased to 10% of the total population in myc-rpt6-S119A.
Fig 2.
Proteasome activity was decreased in the myc-rpt6-S119A mutant.
(A) Lysates from strains in stationary phase (A600>5.0) were subjected to native gel electrophoresis followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC. The myc-rpt6-S119A mutant displays lower activity by native in-gel analysis (before and after 20S gate opening by SDS). (B) Gels from (A) were transferred to nitrocellulose and probed with anti-myc and anti-20S antibodies. Immunoblotting indicated similar levels of proteasomes. Shown are representative in-gel and immunoblot analysis from 3 or more independent experiments. RP = Regulatory Particle, CP = Core Particle. (C) The chymotrypsin-like activity of 26S proteasomes in whole cell lysates of stationary phase cells (A600>5.0) was monitored by Suc-LLVY-AMC cleavage over time on microplate reader (ex. 360nm and em. 465nm). The mean ± SEM rate of Suc-LLVY-AMC cleavage is decreased in the myc-rpt6-S119A mutant compared to the WT and myc-rpt6-S119D strains. *p < 0.05, one way ANOVA. Data are from two biological samples (n = 2) with triplicate analysis.
Fig 3.
Altered Rpt6 phosphorylation and proteasome function exacerbated Htt polyQ aggregation and proteotoxicity.
(A) Representative immunoblot (anti-GFP) of GFP-tagged (Htt) with different polyQ repeats (Htt25Q, Htt72Q and Htt103Q). Expression is similar in all myc-Rpt6 strains. (B) Expression of Htt72Q and Htt103Q increased the sensitivity of myc-rpt6-S119A to canavanine. Strains transformed with URA3 marked vector, Htt25, Htt72 and Htt103 plasmids were plated on canavanine to induce proteotoxic stress. The arg- ura- plates are controls. (C) Representative images of RPT6 mutants transformed with Htt25Q, Htt72Q and Htt103Q constructs. Larger GFP-Htt aggregates are observed in the myc-rpt6-S119A mutant compared to WT and the myc-rpt6-S119D mutant. (D) Quantification of aggregate size (total GFP signal within a cell) is presented for Htt103Q expression. Graphs depict mean aggregate size ± SEM (arbitrary units). **p < 0.01, one way ANOVA. n = 45 to 60 individual GFP-aggregates (cells) measured over 2 independent experiments.
Fig 4.
Senescent myc-rpt6-S119A cells expressing Htt103Q had a delayed loss of viability relative to myc-RPT6 upon chronological aging.
Strains were plated at 30°C to assess viability after 1, 5, 7, 10, 12 and 15 days of growth in liquid medium. Graphs are the average of three independent experiments performed with duplicate samples. Values were plotted relative to myc-RPT6 transformed with vector. Comparison of all strains transformed with p416 control vector (A) or p416-Htt103Q (B). Graph for all strains expressing Htt25Q is shown in S4 Fig.