Journal: Biochemical and biophysical research communications

Article Title: Hsp104 is essential for the selective degradation in yeast of polyglutamine expanded ataxin-1 but not most misfolded proteins generally

doi: 10.1016/j.bbrc.2009.12.018

Figure Lengend Snippet: Mutant human ataxin-1 with expanded polyglutamine repeats (82Q) is selectively degraded by the proteasome in yeast. Yeast cells (JN284) were transfected with the plasmids carrying human ataxin-1 (30Q or 82Q) whose expression is under the control of the GAL1 promoter. Cells were grown in the minimal medium containing 2% raffinose until mid-log phase (OD600 = 0.5–1.0) and then transferred to the induction medium containing 2% galactose for 6–8 h. The degradation of ataxin-1 in these cells was measured in the presence or absence of MG132 (50 µM) either by pulse-chase analysis and immunoprecipitation with the anti-ataxin-1 antibody 11750 or by the promoter shut-off assay. Similar results were obtained in three independent experiments.

Article Snippet: The yeast expression plasmids carrying human ataxin-1 (wild-type and mutant) under the control of GAL1 promoter – provided by Prof. Huda Zoghbi (Baylor College of Medicine) – were constructed by inserting human ATXN1 (SCA1) coding region into SpeI and SalI sites of p416 Gal1 or p423 Gal1 vector (ATCC).

Techniques: Mutagenesis, Transfection, Expressing, Control, Pulse Chase, Immunoprecipitation

Journal: Biochemical and biophysical research communications

Article Title: Hsp104 is essential for the selective degradation in yeast of polyglutamine expanded ataxin-1 but not most misfolded proteins generally

doi: 10.1016/j.bbrc.2009.12.018

Figure Lengend Snippet: Hsp104 is required for the selective degradation of mutant human ataxin-1. (A) Selective degradation of mutant ataxin-1 [82Q] does not require Ydj1p, an Hsp40 homolog, (B) but does require Hsp104. Plasmids carrying the mutant [82Q] ataxin-1 were transfected into (A) the wild-type (W303-1b) and temperature-sensitive mutant strain of Ydj1p (ACY 17b: ydj1–151) or (B) the wild-type (W303a) and Hsp104-deletion mutant strain (YS 483: Δhsp104). Degradation of ataxin-1 was then measured as described in Fig. 1. (C) Transfection of Hsp104 into the Hsp104-deletion strain restores the degradation of mutant ataxin-1 back to the control level. A CEN plasmid carrying the wild-type Hsp104 (under the GAL1 promoter) was introduced into Δhsp104 cells expressing the mutant ataxin-1, and its degradation was measured. (D) Hsp104 deletion does not affect the degradation of the model substrate ubiquitin-Pro-β-galactosidase (Ub-Pro-β-gal).

Article Snippet: The yeast expression plasmids carrying human ataxin-1 (wild-type and mutant) under the control of GAL1 promoter – provided by Prof. Huda Zoghbi (Baylor College of Medicine) – were constructed by inserting human ATXN1 (SCA1) coding region into SpeI and SalI sites of p416 Gal1 or p423 Gal1 vector (ATCC).

Techniques: Mutagenesis, Transfection, Control, Plasmid Preparation, Expressing, Ubiquitin Proteomics