Yeast prions: structure, biology and prion-handling systems Supplementary Information Phenotypes of wild [PSI+] strains. Methods Yeast strains UCD#824, UCD#939 and UCD#978 were purchased directly from UC Davis Department of Viticulture and Enology culture collection. Strains were cured of [PSI+] by selecting three single colonies and growing to single colonies on YPAD media containing 3mM guanidine for three successive plates. The final YPAD 3mM guanidine plate was stamped to YPG to verify that colonies were rho+, which they all were. Wild [PSI+] strains were also transformed with either pdb132 (KanMX CEN vector) or with pdb145 (KanMX CEN ADH promoted Sup35C domain only). This plasmid was generated by digesting pdb10 and pdb132 with BamH1 and Xho1, and ligating the product of the pdb10 digestion into pdb132. G418 Sulfate was purchased from Mediatech Inc. (Herndon, VA, Cat no. 61 234 RG, lot 61234113), tert Butyl hydroperoxide solution (tbooh, Cat no. 458139 100mL, lot MKBP8343V), fluconazole (Cat no. F8929 100MG, lot 102M4731V) and 4 nitroquinoline n oxide (4NQO, Cat no. N8141 1G, lot MKBQ7749V) were purchased from Sigma Aldrich (St.Louis, MO). All plates and yeast media were prepared using standard techniques, YPD ph 4 was prepared by lowering the ph of YPD using drop wise addition of 6N HCl monitored with a Thermo Orion 350 ph meter. Stock 64 mg/ml concentration of fluconazole was prepared by dissolving 100mg in 1.56 ml of 200 proof ethyl alcohol. Stock 4 mg/ml 4NQO was prepared by dissolving 100 mg into 25 ml DMSO. For the concentration series, stock fluconazole was diluted 1:200 in YPD with or without 0.2 mg G418/ ml, while stock 4NQO and tbooh were diluted 1:1000 directly in either YPD or YPD containing 0.2 mg/ml G418; suitable volumes were added to the wells. Strains were grown on YPAD or YPAD containing 0.2 mg/ml G418 plates and diluted in water to a density of 1000 cells/ul. For growth studies 10000 cells were added per well in 96 well Corning Costar clear plates with 150 ul total culture volume. Growth measurements were recorded at 550nm using a Molecular devices SpectroMax M5 plate reader. Data was imported and analyzed using Microsoft Excel 2007. On each plate, every experiment was done in triplicate, and three similar independent experiments were done for each strain condition combination, with similar results, though only one is shown. Invasive growth of strain UCD#978 was tested on YPD agar medium and on SPHD medium (1) with 10 mg G418 per plate in some cases. Cells were streaked for single colonies, incubated for 10 days at 30C, and the plates were placed under running water and rubbed gently. Results Halfmann et al. compared growth of wild [PSI+] strains with guanidine cured derivatives or with the [PSI+] strain transformed with a plasmid expressing Sup35C, the part of the molecule lacking the prion domain (2). They reported a growth advantage for [PSI+] for strain UCD#824 in the presence of 32 mg/ml fluconazole, and for UCD#939 in the presence of 0.4 mg/ml 4NQO (2). Strain UCD#978 was reported to be more sensitive to tbooh in the presence of [PSI+] than in its absence, but showed invasive growth on agar with the prion but not without (2). Strain UCD#824 was reported to grow at ph4 in the presence of [PSI+], but not in its absence (1). We find that at 0.4 mg/ml 4NQO all yeast cells are dead and the control with 10 % DMSO significantly impacts yeast growth rates. Similarly 32 mg/ml flucoazole would present the yeast in 50 % ethanol, which would probably kill all the yeast cells. Thus we decided to use a series of concentrations including 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 ug/ml 4NQO, 0.12, 0.25, 0.5, 1, 2, and 4 mm tbooh, and 4, 8, 16, 32, 64 and 128 ug/ml fluconazole.
We also tested strain UCD#824 in YPD at ph4 and ph7. We did not observe any difference in growth rates for strain UCD#824 between ph4 and ph7 YPD media (Fig. S1, S2), for strain UCD#939 in any concentration of 4NQO (Fig. S5, S6) and no significant difference in growth rates for strain UCD#978 in tbooh (Fig. S7, S8). Nor do we see any difference in invasive growth of strain UCD#978 (Fig. S9). We do see a significant effect at several concentrations of fluconazole: the [PSI+] UCD#824 strain grew better with the vector than with the plasmid expressing only the Sup35C domain, and the [PSI+] modestly better than the cured [psi ] (Fig. S3, S4). References: 1. Gimeno CJ, Ljungdahl PO, Styles CA, Fink GR. 1992. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth regulation by starvation and RAS. Cell 68:1077-1090. 2. Halfmann R, Jarosz DF, Jones SK, Chang A, Lancster AK, Lindquist S. 2012. Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature 482:363-368.
Fig. S1: Wild yeast strain UCD824 containing [PSI+] or cured, display no growth differences in media at ph 4 or ph 7. Data presented is the average results of three independent experiments each performed in triplicate. Fig. S2: Wild yeast strain UCD824 containing either G418 vector or G418 Sup35C domain only. There seems to be no growth difference between media at ph4 or ph7. Data presented is the average results of three independent experiments each performed in triplicate.
Fig. S3: Wild yeast strain UCD824 containing [PSI+] or cured, display little growth differences in a concentration range of fluconazole. Concentrations at and above 32 ug/ml seem toxic to cells. Data presented is the average results of three independent experiments each performed in triplicate. Fig. S4: Wild yeast strain UCD824 containing either G418 vector or G418 Sup35C domain only. There seems to be a slight growth advantage for the vector containing cells at only the 32 ug/ml concentration. Data presented is the average results of three independent experiments each performed in triplicate.
Fig. S5: Wild yeast strain UCD939 containing [PSI+] or cured, display very little growth differences in a concentration range of 4NQO. Concentrations at and above 0.4 ug/ml seem to kill cells. Data presented is the average results of three independent experiments each performed in triplicate. Fig. S6: Wild yeast strain UCD939 containing either G418 vector or G418 Sup35C domain only. Concentrations at and above 0.4 ug/ml 4NQO seems to kill cells. Data presented is the average results of three independent experiments each performed in triplicate.
Fig. S7: Wild yeast strain UCD978 containing [PSI+] or cured, display very little growth differences in a concentration range of t-booh. Concentrations at and above 0.5 mm t- BOOH seem to kill cells. Data presented is the average results of three independent experiments each performed in triplicate. Fig. S8: Wild yeast strain UCD978 containing either G418 vector or G418 Sup35C domain only were grown in YPD liquid with various concentrations of t-booh. Concentrations of t- BOOH at and above 0.5 mm seem to kill cells. Data presented is the average results of three independent experiments each performed in triplicate.
Fig. S9: Invasive growth test of strain UCD#978. Cells were grown for 10 days on SPHD plates (right) and washed with water (left). No difference between [PSI+] and cured [psi-] cells or between [PSI+] with vector and [PSI+] expressing Sup35C was observed.
Examination of reported guanidine-curable phenotypes of wild strains. It is reported that 1/3 of wild yeast not known to have any prions have phenotypes curable by guanidine, suggesting that these strains in fact do carry prions (1). We have reexamined this issue using several of these strains. Several wild strains purported to harbor uncharacterized Hsp104-dependent prions were grown in the presence of 3 mm guanidine hydrochloride for ~60 generations. Using conditions in which the underlying prions were reported to alter phenotypes, the growth of guanidinetreated and untreated strains were compared over 24 hours using a 96-well plate reader. The following strains were grown at 30oC with the indicated supplementation: WE372 (YPD), YJM428 (YPD, 0.5 M NaCl), YJM653 (YPD, 0.5 M NaCl) and I14 (YPD, 50 ug/ml fluconazole). No significant differences were observed (Fig. S10). 1. Halfmann R, Jarosz DF, Jones SK, Chang A, Lancster AK, Lindquist S. 2012. Prions are a common mechanism for phenotypic inheritance in wild yeasts. Nature 482:363-368.
Fig. S10. Re examination of guanidinecurable phenotypes of wild strains. Expected Phenotypic Change According to Halfmann et al. Nature 2012. 4 Strain WE372 on YPD Absorbance 3 2 1 0 0 5 10 15 20 Hours WE372 WE372 G Decreased growth on YPD following guanidine treatment (G) Absorbance 4 3 2 1 0 Strain YJM428 on NaCl 0 5 10 15 20 Hours YJM 428 YJM 428 G Decreased growth on high NaCl following guanidine treatment (G) Absorbance 4 3 2 1 0 Strain YJM653 on NaCl 0 5 10 15 20 Hours YJM653 YJM653 3G Decreased growth on high NaCl following guanidine treatment (G) Absorbance 4 3 2 1 0 Strain I14 on fluconazole 0 5 10 15 20 Hours I14 I14 G Decreased growth on fluconazole following guanidine treatment (G)