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Supporting Materials and Methods

Strain Construction. Yeast media and standard cultivation procedures are described in refs. 1 and 2). The yeast strains used are all derivatives of the original [psi-][RNQ+] 74-D694 (MATa ade1-14 his3-D 200 ura3-52 leu2-3,112 trp1-289) (3). [psi-][rnq-] (1G4) was obtained from [psi-][RNQ+] (1Y1) by GuHCl treatment, [PSI+][RNQ+] from [psi-][RNQ+] by Sup35 overproduction, and [PSI+][rnq-] from [PSI+][RNQ+] by GuHCl treatment (4-6). Pairs of [PSI+] derivatives carrying either strong or weak [PSI+] variants (4) were used. The [RNQ+] variant in all derivatives is a high [PIN+] (7).

Primers and Plasmid Construction. YFP and CFP were PCR-amplified from pDH3 and pDH5 (YRC, http://depts.washington.edu/~yeastrc; with primers 5'-gtccccgcggagtaaaggagaacttttc and 5'-tccgagctctcatttgtatagttcatccatgcc, and SacI/SacII-digested PCR fragments were cloned to replace GFP. Synph-1 (amplified by using primers 5'-ggactagtatggaagcccctgaataccttg' and 5'-ggggaagctttgctgccttattctttcctttgc'; cloned as a SpeI/HindIII-digested PCR fragment) was fused to YFP (amplified by using primers 5'-cgatcgatagtaaaggagaagaacttttc' and 5'-ggtctcgagttattatttgtatagttcatccatgcc'; cloned as a ClaI/XhoI-digested PCR fragment) in a LEU2 vector. TTR-WT and TTRd lacking the region encoding the signal sequence (amplified by using primers 5'-cggggatccatgggtcctacgggcaccgg' and 5'-ggtaagcttttccttgggattggtgacg'; cloned as BamHI/HindIII PCR fragment) were fused to CFP (amplified by using primers 5'-cgatcgatagtaaaggagaagaacttttc' and 5'-ggtctcgagttattatttgtatagttcatccatgcc'; cloned as a ClaI/XhoI-digested PCR fragment) in a URA3 vector.

Microscopy. For most cells (e.g. Table 3), fluorescence of CFP and YFP was analyzed by using a Zeiss Axioskop 2 fluorescent microscope fitted with a Zeiss AxioCam digital camera. Multiple focal planes (Z-stacks) were analyzed for at least 20 cells by using motorized Axiovert 200 or Axioplan II fluorescent Zeiss microscopes and were processed by using Zeiss AxioVision or Huygens Essential software.

Protein Purification. NM was purified as described (8). Recombinant, methanol-precipitated Rnq1, provided by N. Sondheimer (University of Chicago), was dissolved in 4 M Urea, 1´ PBS, pH 7.4, to 5 m M and converted to amyloid fibers by rotation at 40 rpm for several days at room temperature (9). Aggregated Rnq1 was sedimented, washed ,and resuspended in an equal volume of 1´ PBS, pH 7.4. Bovine pancreas insulin (Sigma, catalog no. I6634) was dissolved in 0.01 M HCl to 10 mg/ml and converted into amyloid fibers by sequential heating and cooling (10). Rnq1 and insulin amyloid formation was verified by atomic force microscopy (8). Ig light-chain k (SMA) amyloid was a gift of K. Sciarretta and Y. Argon (University of Chicago). TTR-WT and a Syn amyloids were kindly provided by J. W. Kelly (Scripps Research Institute, La Jolla, CA). Chicken egg white lysozyme (Sigma, catalog no. L6876) was dissolved in 20 mM Tris HCl, pH 7.4/150 mM NaCl/1 mM EDTA to 0.1 mg/ml, and aggregated upon the addition of DTT to 10 mM. Bovine pancreas RNase A (Roche, catalog no.109169) was dissolved in 1´ PBS, pH 7.4, to 1 mg/ml. Solutions of Rnq1, Ig, and insulin preformed fibers were sonicated twice for 15 sec at 30-sec intervals at 4°C with a Heat Systems Ultrasonics cell disruptor (model W-225R) fitted with a microtip. Sonication was required to suspend Rnq1 fibers.

 

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