Suppression of Rnq1 toxicity by Sis1 correlates with the nuclear accumulation of Rnq1 aggregates. (A) Sytox dye uptake into W303 [RNQ⁺] cells expressing Rnq1 with and without excess Sis1. Rnq1 overexpression was induced for 5 h before the addition of Sytox dye, at which point fluorescent readings were taken every min for 30 min. (B) Rnq1-mRFP localization in the nucleus of YEF473B [RNQ⁺] cells resultant from Sis1 overexpression. Fluorescent images show the cellular distribution of Rnq1-mRFP, which was overexpressed for 15 h from the CUP1 promoter due to the presence of 10 μM CuSO₄ in media. Nup49-GFP is a marker of the nuclear envelope boundary. DAPI denotes the localization of nuclear DNA. (C) Effect of chaperone overexpression on the cellular distribution of Rnq1-GFP in W303 [RNQ⁺] cells. Fluorescent images show the cellular distribution of Rnq1-GFP, which was overexpressed for 15 h from the CUP1 promoter due to 1 μM CuSO₄ present in media. The GFP panel on the right shows the localization of GFP under conditions describes on the left. (D) Western blot analysis of the indicated proteins from cell extracts in C.

Rnq1-GFP-NLS is less toxic than Rnq1-GFP in W303 [RNQ⁺] cells. (A) Analysis of toxicity in fivefold serial dilutions of W303 [RNQ⁺] cells grown on plates supplemented with galactose to induce Rnq1 expression from the GAL1 promoter. (B) Localization of Rnq1-GFP-NLS and Rnq1-GFP via fluorescence microscopy. Fixed cells were permeabilized and stained with DAPI in the merge. (C) SDD-AGE analysis of the time course for assembly of SDS-resistant Rnq1-GFP aggregates after induction of Rnq1-GFP expression with 2% galactose. (D) Filter trap analysis of Rnq1-GFP conversion to an SDS-resistant species at the indicated time after induced expression. Rnq1-GFP retained on the cellulose acetate filter was detected with α-GFP. In quantitations, Rnq1-GFP-NLS band intensity was compared with the Rnq1-GFP signal at the 90-min time point. (E) Time course of Rnq1-GFP expression determined by Western blot. (F) Populations of exogenous Rnq1-GFP and endogenous Rnq1 species were determined by size exclusion chromatography 4 h after the induced expression of either Rnq1-GFP or Rnq1-GFP-NLS via supplementation of media with 2% galactose. Input corresponds to 10% of the total lysate protein loaded. Quantitation of signal intensity in each peak was compared with the total signal from the sum of all fractions.

Expression of Rnq1-GFP-NLS promotes the nuclear accumulation of Rnq1-mRFP and suppresses Rnq1 L94A toxicity in W303 [RNQ⁺] cells. (A) Prior expression of Rnq1-GFP-NLS leads to the accumulation of Rnq1-mRFP in the nucleus. Rnq1-GFP and Rnq1-GFP-NLS were expressed for 15 h at low, nontoxic levels from the CUP1 promoter due to the presence of 1 μM CuSO₄ in media. Subsequent expression of Rnq1-mRFP from the GAL1 promoter was induced by the addition of 2% galactose. After 1 h, the location of the GFP and mRFP signals was determined in fixed, DAPI-stained cells by fluorescence microscopy. (B) Analysis of cell growth in fivefold serial dilutions of W303 [RNQ⁺] cells that harbored the indicated constructs. Cells were grown overnight in synthetic media containing 1 μM CuSO₄ to allow for Rnq1-GFP or Rnq1-GFP-NLS expression from the CUP1 promoter. Cells were then plated on agar that contained glucose (the control) or galactose to induce Rnq1-GFP L94A expression to toxic levels. (C) Rnq1-GFP-NLS in the nucleus of cytoduced W303a [RNQ⁺] cells sequestered Rnq1-mRFP from the cytosol of 10B-H49α [rnq-] cells. Rnq1-GFP-NLS was expressed from the GAL1 promoter and Rnq1-mRFP expression was under control of the CUP1 promoter. Two hours before mixture of different mating types, W303 and 10B-H49 cell cultures harboring the indicated Rnq1 constructs, were supplemented with 2% galactose and 50 μM CuSO₄, respectively. Cell fusion was analyzed 30 min (top row, control) and 2 h (bottom row) after cell mixture. Merge images represent the combination of all fluorescent channels.

Htt-103Q-GFP-NLS forms SDS-resistant aggregates with reduced efficiency and is dramatically more toxic than Htt-103Q-GFP in W303 [RNQ⁺] cells. (A) Analysis of cell growth in fivefold serial dilutions of W303 [RNQ⁺] and [rnq-] cells harboring the indicated forms of Htt-103Q that were expressed from the GAL1 promoter. (B) Filter trap analysis of SDS-resistant Htt-103Q and Htt-103Q-GFP-NLS aggregates. Htt-103Q chimeras were overexpressed for 4 h in W303 [RNQ⁺] and [rnq-] cells. Bottom panels are Western blots of cell extracts used in the filter trap assay. In quantitations, Htt-103Q-NLS band intensity was compared with the Htt-103Q signal from [RNQ⁺] cell extracts. (C) Cellular localization of Htt-103Q-GFP and Htt-103Q-GFP-NLS in W303 [RNQ⁺] cells as determined by fluorescence microscopy. The expression of Htt fusion proteins was driven by the addition of 2% galactose for 4 h before fluorescent images were obtained. (D) Comparison of Htt-103Q-GFP and Htt-103Q-GFP-NLS assembly status in W303 [RNQ⁺] cells by size exclusion chromatography. Htt-103Q chimeras were expressed for 2 h in cells harboring only endogenous Rnq1.

The impact of the order of Sis1 and Rnq1-GFP overexpression on Rnq1-GFP localization (A) The localization of Sis1-GFP in BY4741 [RNQ⁺] cells was monitored by fluorescence microscopy. Fixed cells were permeabilized and stained with DAPI in the merge. (B) Cofractionation of Sis1 with nuclei in cell extracts. Differential centrifugation was performed on native cell extracts from W303 [RNQ⁺] cells to determine the amount of Sis1 that could pellet with the nuclear marker Nop1 after centrifugation at 13,000 × g. Western blot analysis shows the levels of the indicated protein in the total (T), supernatant (S), or pellet (P) fractions. Sis1 was overexpressed from the constitutively active GPD promoter. Untagged Rnq1 was overexpressed from the GAL1 promoter via addition of 2% galactose to cell cultures. (C) Fluorescent images show the cellular distribution of Rnq1-GFP that was expressed from the CUP1 promoter at low, nontoxic levels due to the presence of 1 μM CuSO₄ in media. Sis1 was overexpressed from the constitutively active GPD promoter. Rnq1-GFP and Sis1 overexpression constructs were individually introduced into W303 [RNQ⁺] cells via sequential transformations, and the order of transformation of each protein was designated as 1st or 2nd. Rnq1-GFP fluorescence was overlaid onto phase images. (D) Quantitation of Rnq1-GFP nuclear compartmentalized populations of 200 cells from three separate transformations. Values are expressed in percentage of total cells counted and the error bars reflect the SD. The p value was <0.0001. (E) Expression levels of the indicated proteins by Western blot analysis.

Targeting Rnq1-GFP to the nucleus is toxic in W303 [rnq-] cells. (A) Fivefold serial dilutions of W303 [rnq-] cells harboring the indicated Rnq1-GFP proteins were grown on agarose plates containing galactose. (B) The ability of different Rnq1-GFP proteins to assemble into SDS-resistant aggregates was monitored in [rnq-] cells by SDD-AGE analysis. The left lane shows Rnq1-GFP from W303 [RNQ⁺] cell extracts. (C) Fluorescent distribution of different Rnq1-GFP proteins whose expression was driven by 2% galactose for 4 h in [rnq-] cells. Fixed cells were permeabilized and stained with DAPI in the merge image.

The presence of Rnq1-GFP aggregates in the nucleus of W303 [RNQ⁺] cells exacerbates Htt-103Q toxicity. (A) Analysis of cell growth in fivefold serial dilutions of [RNQ⁺] cells, which harbored the indicated constructs. Rnq1-GFP and Rnq1-GFP-NLS were first expressed for 15 h at nontoxic levels from a CUP1 promoter due to the presence of 1 μM CuSO₄ in the culture. Then, Htt-25Q-GFP and Htt-103Q-GFP were expressed at toxic levels from the GAL1 promoter on agarose plates containing 2% galactose. (B) The impact of Rnq1-mRFP-NLS expression on the cellular localization of Htt-25Q-GFP and Htt-103Q-GFP. Rnq1-mRFP or Rnq1-mRFP-NLS was expressed for 15 h at low nontoxic levels from the CUP1 promoter due to the presence of 10 μM CuSO₄ in media. Subsequent expression of Htt-25Q-GFP and Htt-103Q-GFP from the GAL1 promoter was induced by the addition of 2% galactose. After 1 h, the location of the GFP and mRFP signals was determined in fixed, DAPI-stained cells by fluorescence microscopy. (C) Coprecipitation of endogenous Rnq1 and Htt-103Q-GFP in complex with each other. Extracts were generated from W303 [RNQ⁺] cells expressing Htt-103Q-GFP and endogenous Rnq1 was precipitated under native conditions with α-Rnq1. Precipitates were resolved by SDS-PAGE and Western blots were probed with α-GFP or α-Rnq1 to detect Htt-103Q-GFP or endogenous Rnq1, respectively. (D) Coprecipitation of Htt-103Q-GFP in complex with Rnq1-GFP and Rnq1-GFP-NLS. Nondenaturing cell extracts were generated from W303 [RNQ⁺] cells. Rnq1-GFP and Rnq1-GFP-NLS were precipitated by the addition α-Rnq1 and Western blots were probed with α-GFP (bottom) or α-FLAG (top) to detect the different forms of Rnq1 and Htt. (E) Filter trap analysis of SDS-resistant Htt-103Q aggregates. Htt-103Q retained on the cellulose acetate filter was detected with α-FLAG. Bottom panels are western blots of cell extracts used in the filter trap assay. In quantitations, Htt-103Q band intensity from cells expressing Rnq1-GFP and Rnq1-GFP-NLS was compared with the Htt-103Q signal in the absence of exogenous Rnq1. (F) Analysis of Htt-103Q assembly status in W303 [RNQ⁺] cells. Rnq1-GFP or Rnq1-GFP-NLS under control of the CUP1 promoter was expressed for 15 h at low nontoxic levels by the presence of 1 μM CuSO₄ in cell cultures. Then Htt-103Q was expressed for 2 h by the addition of 2% galactose. Proteins in cell extracts were resolved by size exclusion chromatography. Top gels in each set of panels show the quantity of SDS-resistant Htt-103Q that was incapable of gel entry. The second set of panels from the top shows Htt-103Q-GFP which was able to migrate into gels. The third set of panels from the top shows the mobility of either Rnq1-GFP or Rnq1-GFP-NLS. The bottom set of panels shows the mobility of endogenous Rnq1. Input represents 10% of the total amount of protein detected in extracts. Quantitation of Htt-103Q signal intensity in each peak was compared with the total signal from the sum of all fractions.