Figure 1.
High copy plasmids that enhance induction of [PSI+] also cure pre-existing [PSI+] and [URE3].
(A) Overexpressed Q/N-rich proteins cause loss of [PSI+]. Weak (w) [PSI+][PIN+] (L1758) was transformed with plasmids encoding the indicated Q/N-rich proteins or fragments (PIN4C and CYC8C), 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 [psi−] among ∼1100 colonies. (B) Induction of [PSI+] in a rnq1Δ::HIS3 [psi−][pin−] 74-D694 strain (L3125) carrying the same plasmids as in (A). [PSI+] was induced by overexpression of SUP35NM-GFP from pCUP1-SUP35NM::GFP-TRP1 in 50 µM Cu2+ following library plasmid amplification on SD-Leu. Shown is growth on SD-Ade, which indicates the presence of [PSI+]: spots are representative of three repeated experiments. The Ade+ colonies were verified to be [PSI+] by visualization of Sup35NM-GFP dots. (C) Overexpressed Q/N-rich domains cause [URE3] curing. The [URE3] derivative of 74D-694 [PIN+][psi−] 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 [ure-o] cells among ∼1000 colonies was determined using the color assay described in Materials and Methods. Error bars show standard error of the mean.
Figure 2.
Pin4C overexpression leads to larger [PSI+] aggregates.
(A) Sup35-GFP aggregates become larger upon overexpression of Pin4C. Upper panels: Representative GFP images of strong [PSI+][PIN+] SUP35-GFP expressing strains (GF657) after an overnight induction of PIN4C; empty vector control was incubated in galactose medium for the same amount of time; [psi−] culture shown is after 4-days of induction of PIN4C. Lower panels: Lysates prepared from cultures shown above were treated with 2% SDS at room temperature and analyzed for the presence of Sup35 by SDD-AGE with anti-Sup35C-GFP. (B) Overexpression of Pin4C-DsRed leads to the formation of single dot-like aggregates concomitant with enlargement of Sup35-GFP aggregates. Representative DsRed and GFP images are of strong [PSI+][PIN+] SUP35-GFP (GF657) cells after induction of pHR81GAL-PIN4C-DsRED for the times indicated. Cells were grown in liquid plasmid selective galactose media. (C) Pin4C-DsRed aggregates are composed of SDS-resistant polymers. Lysates from cultures where ∼60% of cells contain large single Pin4-DsRed dots were treated with 2% SDS at room temperature and analyzed by SDD-AGE. The blot was probed with anti-DsRed antibody (left lane), stripped and re-probed with anti-Sup35C antibody (right lane). (D) Increased size of visible [PSI+] aggregates requires continuous synthesis of the Sup35 protein. Single cells of strong [PSI+][PIN+] expressing SUP35ΔNM at its endogenous locus and harboring extrachromosomal pTETr-SUP35-GFP (L3126) were transformed with pHR81GAL-PIN4C-DsRED, or vector, pHR81GAL-DsRED. Cells were grown in 2% raffinose +2% galactose +0.025 µg/ml doxcycline for 6 hrs, which induced PIN4C-DsRED and allowed the Sup35-GFP level to be close to the normal Sup35 level. Single cells with diffuse DsRed were then micromanipulated and grown for 18 hrs dividing ∼3 times on 2% raffinose +2% galactose +10 µg/ml doxcycline medium where new synthesis of Sup35-GFP is repressed. GFP and DsRed images of a representative part of a growing microcolony are shown. The arrows point to mother cells, and the arrowheads point to two daughter cells with and without a large DsRed dot.
Figure 3.
Overexpressed Pin4C reduces the transmission of Sup35-GFP from mother to daughter cells.
(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 [70]. (B–D) Quantitative FRAP analysis of Sup35-GFP in 10 [psi−][pin−] (GF658) cells (B), 9 strong [PSI+][PIN+] (GF657) cells harboring the pHR81GAL vector (C), and 12 strong [PSI+][PIN+] (GF657) cells harboring pHR81GAL-PIN4C 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.
Figure 4.
Microcolonies overexpressing Pin4C-DsRed show progressive loss of [PSI+].
Single strong [PSI+][PIN+] SUP35-GFP expressing (GF657) cells carrying pHR81GAL-PIN4C-DsRED 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.
Figure 5.
Curing of [PSI+] by Pin4C depends on cell division.
Three MATa strong [PSI+][PIN+] SUP35-GFP (GF845) transformants harboring pHR81GAL-PIN4C were grown in liquid galactose media for 40 hrs, when Sup35-GFP foci became larger in size and fewer in number. Then cells were transferred to fresh galactose medium with or without the addition of 50 µM a-factor for another 16 hrs. Three transformants harboring empty vector pHR81-GAL were transferred to galactose medium without the addition of 50 µM a-factor as control. Samples were taken, diluted and plated on YPD where the percentage of red cured [PSI+] among ∼850 colonies was scored (black bars). There was no [PSI+] loss in the control. The number of CFUs is shown by gray bars. Error bars show standard error of the mean.
Figure 6.
Overexpressed Pin4C sequesters Hsp104 from the cytoplasm.
(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 pHR81GAL-PIN4C-DsRED are shown. (B) Overexpressed Pin4C binds to Hsp104. The interaction between Hsp104 and overexpressed Pin4C was assayed in strong [PSI+][PIN+] (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 [PSI+] by Pin4C. Transformants with pHR81GAL-PIN4C and pRS413GAL-HSP104T160M (↑Pin4C, ↑Hsp104T160M); or with pHR81GAL-PIN4C and empty vector pRS413GAL (↑Pin4C); or with pRS413GAL-HSP104T160M and pHR81GAL (↑Hsp104T160M); or with both empty vectors pHR81GAL and pRS413GAL (↑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 Hsp104T160M is turned off. There was no growth inhibition in cells overexpressing Pin4C and Hsp104T160M compared to those overexpressing Pin4C alone when spotted on a galactose plate (Figure S5). [PSI+] loss was scored by the appearance of red [psi−] colonies.
Figure 7.
Effects of overexpressed Pin3 and Cyc8C on Hsp104.
(A) Overexpressed Pin3 caused endogenous Hsp104-GFP to coalesce into big aggregates. Representative images of Hsp104-GFP cells after induction of pHR81GAL-PIN4C or pHR81GAL-PIN3 for 16 hrs are shown. All the images shown were taken in the same exposure time. (B) Hsp104-GFP became brighter upon overexpression of Cyc8C. Representative images of Hsp104-GFP cells after 16 hrs induction of Cyc8C from pHR81 based high copy vectors with the insert encoding CYC8C or the empty vector on SD-Leu liquid medium. All the images shown were taken in the same exposure time. (C) Overexpressed Pin3 caused the sequestration of Hsp104 less effectively than Pin4C. The bar graphs represent the average of the mean fluorescence signal intensity in cytoplasmic regions devoid of aggregates and excluding the vacuole in 22 Hsp104-GFP cells. Error bars indicate the standard error of the mean. P<0.0001 for comparisons of fluorescence intensity in cells with overexpressed Pin4C or Pin3 and vector control.
Figure 8.
Overexpressed Pin4C or Pin3 caused Sis1 to coalesce.
(A) Overexpressed Pin4C or Pin3 causes Sis1-GFP to aggregate. Representative images of endogenous Sis1 tagged with GFP in the absence or presence of 16 hrs of induction of pHR81GAL-PIN4C-DsRED or pHR81GAL-PIN3 are shown. Fixed cells were permeabilized and stained with DAPI. The arrowhead points to nuclear Sis1-GFP signals, and the arrow points to aggregated Sis1-GFP that is not nuclear. (B) Overexpressed Pin3 did not cause effective sequestration of Sis1 from the cytoplasm. The bar graphs represent the average of the mean fluorescence signal intensity in cytoplasmic regions devoid of aggregates and excluding the vacuole in 20 Sis1-GFP cells. Error bars indicate the standard error of the mean. P<0.0001 for comparisons of fluorescence intensity in cells with overexpressed Pin4C or Pin3 and vector control.
Figure 9.
Overexpressed Sis1 reduces elimination of [PSI+] by excess Pin4C.
(A) Increased levels of Sis1 inhibit [PSI+] curing by excess Pin4C. Strong [PSI+][PIN+] (GF657) cells co-transformed with pHR81GAL-PIN4C and pYES3GAL-SISI (↑Pin4C, ↑Sis1); or with pHR81GAL-PIN4C and empty vector pYES3GAL (↑Pin4C); or with pYES3GAL-SIS1 and empty vector pHR81GAL (↑Sis1); or with two empty vectors pHR81GAL and pYES3GAL (↑vectors), were examined as described above (see Figure 6C). (B) Overexpressed Sis1 prevents overproduced Pin4C from forming large aggregates. Representative fluorescent images of strong [PSI+][PIN+] (GF657) cells harboring pHR81GAL-PIN4C-DsRED and pYES3GAL-SISI (↑Pin4C, ↑Sis1), or pHR81GAL-PIN4C-DsRED and empty vector pYES3GAL (↑Pin4C) after overnight induction in liquid galactose are shown.
Figure 10.
Overexpression of the non-Q/N-rich protein Gpg1 leads to larger [PSI +] aggregates and causes Hsp104 to aggregate.
(A) Overexpressed Gpg1 leads to larger [PSI+] aggregates. Representative GFP images of strong [PSI+][PIN+] SUP35-GFP expressing cells (GF657) transformed with pRS316-GAL1-GPG1 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-GAL1-GPG1 or empty vector as control are shown.
Table 1.
Yeast strains.