Centrifugation assay demonstrates aggregation of exogenous human LamR in the presence of the [<i>PSI</i>⁺] prion.

<p>(A) Total lysate (T), supernatant (S), and resuspended pellet (P) (20 µg per sample) of pCUP1-LamR::GFP and pCUP1-LamR transformants of yeast [<i>psi</i>⁻][<i>pin</i>⁻] and [<i>PSI⁺</i>][<i>PIN⁺</i>] strains were analyzed by western blot using the indicated antibodies. pCUP1-GFP was expressed in the [<i>PSI</i>⁺] strain as a control (bottom panel), and anti-yeast hexokinase antibody (anti-HK) was used to ensure pellets were free of cytoplasmic proteins (fifth panel from top). Shown are representative experiments out of 3 independent experiments. (B) Corresponding densitometric quantitation of percent distribution between supernatant and pellet fractions was determined from three independent experiments. Bars show standard error of the mean.</p

LamR co-localizes with visible Sup35 prion aggregates in yeast cells.

<p>pSUP35-GFP and pLamR-mCherry were co-expressed in weak [<i>PSI⁺</i>][<i>pin</i>⁻] and [<i>psi</i>⁻][<i>PIN⁺</i>] cells. Panels show GFP and mCherry fluorescence and their merged images. Brightfield (BF) images of the cells are shown in the far right panel. Two sets of images (A and B) are shown from independent transformants. Images were taken using a100× oil immersion lens. The images were visualized and merged using Adobe photoshop CS4.</p

Immunofluorescence of LamR-GFP and Sup35-GFP fusion proteins reveals aggregation of LamR in [<i>PSI</i>⁺] cells.

<p>Late-log cultures of (A) [<i>PSI⁺</i>][<i>PIN⁺</i>], (B) [<i>PSI⁺</i>][<i>pin</i>⁻], (C) [<i>psi</i>⁻][<i>PIN⁺</i>] and (D) [<i>psi</i>⁻ ][<i>pin</i>⁻] were examined with 100× oil immersion lens of a fluorescent microscope using a 488_ex, 507_em filter. Representative images from two independent transformants of each yeast strain are displayed.</p

Human LamR is expressed in yeast cells.

<p>A. LamR protein (right panel) or LamR-GFP fusion (left panel) expressed in the [<i>PSI</i>⁺][<i>PIN⁺</i>] yeast prion strain grown in synthetic media either supplemented with 25 µM CuSO₄ (right panel and lane 2 of left panel) or containing no excess copper (lane1 of left panel). B. Expression of GPF (27 kDa), and LamR-GFP and Sup35-GFP fusion proteins (64 kDa and 104 kDa, respectively) in [<i>psi</i>⁻][<i>PIN⁺</i>] (−) and [<i>PSI⁺</i>][<i>PIN⁺</i>] (+) yeast strains. Anti-LamR (A) and anti-GFP (B) antibodies were used to detect LamR expression in yeast lysates (25 ug). Numbers in the middle (A) and right (B) refer to protein size markers (kDa). Similar expression levels were observed in [<i>pin</i>⁻] strains (not shown).</p

Overexpression of the non-Q/N-rich protein Gpg1 leads to larger [<i>PSI</i>⁺] aggregates and causes Hsp104 to aggregate.

<p>(A) Overexpressed Gpg1 leads to larger [<i>PSI</i>⁺] aggregates. Representative GFP images of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] <i>SUP35-GFP</i> expressing cells (GF657) transformed with pRS316-<i>GAL1-GPG1</i> or empty vector control were of cells incubated overnight in galactose medium for the same amount of time. (B) Overexpressed Gpg1 caused the aggregation of Hsp104-GFP. Representative images of cells with endogenous Hsp104 tagged with GFP with 16 hrs of induction of pRS316-<i>GAL1-GPG1</i> or empty vector as control are shown.</p

Overexpressed Pin4C sequesters Hsp104 from the cytoplasm.

<p>(A) Overexpressed Pin4C sequestered Hsp104-GFP to colocalize with Pin4C-DsRed aggregates. Representative images of cells with endogenous Hsp104 tagged with GFP without or with 16 hrs of induction of pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> are shown. (B) Overexpressed Pin4C binds to Hsp104. The interaction between Hsp104 and overexpressed Pin4C was assayed in strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) following overnight overexpression of Pin4C-DsRed. 60 µg of total protein was loaded as the “lysate”. The Pin4C-DsRed complex immunocaptured with anti-DsRed from 500 µg of total protein was loaded as “eluate”. The same membrane was immunoblotted (IB) with anti-DsRed, then with anti-Hsp104, and re-probed with anti-Pgk1 as a control. No co-immunocapture of endogenous Pgk1 with Pin4C was detected, implying that Hsp104 was specifically immunocaptured with the Pin4C complex. The slightly slower migration of Hsp104 in the “eluate” relative to its migration in the “lysate” is probably due to the different buffers used during immunocapture. (C) Overexpression of Hsp104 ^T160M suppresses curing of strong [<i>PSI</i>⁺] by Pin4C. Transformants with pHR81<i>GAL</i>-<i>PIN4C</i> and pRS413<i>GAL</i>-<i>HSP104^T160M</i> (↑Pin4C, ↑Hsp104^T160M); or with pHR81<i>GAL</i>-<i>PIN4C</i> and empty vector pRS413<i>GAL</i> (↑Pin4C); or with pRS413<i>GAL</i>-<i>HSP104^T160M</i> and pHR81<i>GAL</i> (↑Hsp104^T160M); or with both empty vectors pHR81<i>GAL</i> and pRS413<i>GAL</i> (↑vectors) were selected on plasmid selective glucose medium, replica-plated to plasmid selective inducing galactose medium, and then 10-fold serially diluted (10⁵→10⁰ cells from left to right) and spotted onto glucose YPD medium where expression of Pin4C and Hsp104^T160M is turned off. There was no growth inhibition in cells overexpressing Pin4C and Hsp104^T160M compared to those overexpressing Pin4C alone when spotted on a galactose plate (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#pgen.1003236.s005" target="_blank">Figure S5</a>). [<i>PSI⁺</i>] loss was scored by the appearance of red [<i>psi</i>⁻] colonies.</p

Overexpressed Sis1 reduces elimination of [<i>PSI</i>⁺] by excess Pin4C.

<p>(A) Increased levels of Sis1 inhibit [<i>PSI⁺</i>] curing by excess Pin4C. Strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells co-transformed with pHR81<i>GAL</i>-<i>PIN4C</i> and pYES3<i>GAL</i>-<i>SISI</i> (↑Pin4C, ↑Sis1); or with pHR81<i>GAL</i>-<i>PIN4C</i> and empty vector pYES3<i>GAL</i> (↑Pin4C); or with pYES3<i>GAL</i>-<i>SIS1</i> and empty vector pHR81<i>GAL</i> (↑Sis1); or with two empty vectors pHR81<i>GAL</i> and pYES3<i>GAL</i> (↑vectors), were examined as described above (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#pgen-1003236-g006" target="_blank">Figure 6C</a>). (B) Overexpressed Sis1 prevents overproduced Pin4C from forming large aggregates. Representative fluorescent images of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells harboring pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> and pYES3<i>GAL</i>-<i>SISI</i> (↑Pin4C, ↑Sis1), or pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> and empty vector pYES3<i>GAL</i> (↑Pin4C) after overnight induction in liquid galactose are shown.</p

Microcolonies overexpressing Pin4C-DsRed show progressive loss of [<i>PSI</i>⁺].

<p>Single strong [<i>PSI</i>⁺][<i>PIN</i>⁺] <i>SUP35-GFP</i> expressing (GF657) cells carrying pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> were micromanipulated and grown on 2% raffinose +2% galactose to induce Pin4C-DsRed for ∼24 hrs. Portions of a microcolony are shown as the merge of GFP and DsRed images.</p

Overexpressed Pin4C reduces the transmission of Sup35-GFP from mother to daughter cells.

<p>(A) Diagram of experiment. Fluorescence in daughter cells was photobleached and the time course of fluorescence recovery of the daughter cells was measured as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#pgen.1003236-KawaiNoma1" target="_blank">[70]</a>. (B–D) Quantitative FRAP analysis of Sup35-GFP in 10 [<i>psi</i>⁻][<i>pin</i>⁻] (GF658) cells (B), 9 strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells harboring the pHR81GAL vector (C), and 12 strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells harboring pHR81GAL-<i>PIN4C</i> and containing enlarged Sup35-GFP foci following overnight Pin4C overexpression (D). The relative fluorescence intensity (RFI) of the bleached daughter cell was determined every 5 s after completion of photobleaching and normalization. Error bars indicate the standard error of the mean. RFI in (D) represents the average of 3 cells (I), 4 cells (II) and 3 cells (III). The analysis of two more cells is not shown. (E) The recovery plateau level and half-time from the curves in B–D are listed.</p

High copy plasmids that enhance induction of [<i>PSI</i>⁺] also cure pre-existing [<i>PSI</i>⁺] and [<i>URE3</i>].

<p>(A) Overexpressed Q/N-rich proteins cause loss of [<i>PSI</i>⁺]. Weak (w) [<i>PSI</i>⁺][<i>PIN</i>⁺] (L1758) was transformed with plasmids encoding the indicated Q/N-rich proteins or fragments (<i>PIN4C</i> and <i>CYC8C</i>), or with the empty vector pHR81. Representative images of transformants plated on YPD following amplification of plasmids on SD-Leu are shown (upper). The efficiency of curing (lower) was determined as the percentage of red colonies indicative of [<i>psi</i>⁻] among ∼1100 colonies. (B) Induction of [<i>PSI</i>⁺] in a <i>rnq1Δ</i>::<i>HIS3</i> [<i>psi</i>⁻][<i>pin</i>⁻] 74-D694 strain (L3125) carrying the same plasmids as in (A). [<i>PSI</i>⁺] was induced by overexpression of <i>SUP35NM-GFP</i> from p<i>CUP1</i>-<i>SUP35NM::GFP-TRP1</i> in 50 µM Cu²⁺ following library plasmid amplification on SD-Leu. Shown is growth on SD-Ade, which indicates the presence of [<i>PSI</i>⁺]: spots are representative of three repeated experiments. The Ade⁺ colonies were verified to be [<i>PSI</i>⁺] by visualization of Sup35NM-GFP dots. (C) Overexpressed Q/N-rich domains cause [<i>URE3</i>] curing. The [<i>URE3</i>] derivative of 74D-694 [<i>PIN</i>⁺][<i>psi</i>⁻] expressing the GFP tagged endogenous Sup35 (L3154), was transformed with high copy plasmids encoding the Q/N-rich proteins or protein fragments, or with the empty vector pHR81. The percentage of cured [<i>ure-o</i>] cells among ∼1000 colonies was determined using the color assay described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#s4" target="_blank"><i>Materials and Methods</i></a>. Error bars show standard error of the mean.</p