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Figure 5 Analysis of aggregation of chimeric or truncated Sup35 variants in [PSI+] cells of 74-D694 (ade1-14 sup35::TRP1). (A) State of Sup35SC-BFP and Sup35SC-segment-GFP aggregation visualized by double fluorescence microscopy. [PSI+] cells doubly transformed with pRS313 (HIS3 marker) and pRS316 (URA3 marker) derivatives carrying the indicated fusions were examined. Both fusions were constitutively expressed from the TPI promoter. (B) In vivo kinetics of homotypic and heterotypic Sup35 aggregation. [PSI+] cells were doubly transformed with pRS313 carrying NMSSS-BFP (under the control of the TPI promoter) and pRS316 carrying chimeric NM fusions to GFP (under the control of the CUP1 promoter): a, NMSSS; b, NMSKK; c, NMKSK; d, NMKKK. Upon induction by adding CuSO4 to the growing culture, cells were subjected at 1 h intervals to fluorescent microscopic and immunoblotting analyses. The curves represent the fraction (%) of BFP-foci cells that possessed GFP foci. In these cells, GFP foci were always colocalized with BFP foci. Inserts show the immunoblots of GFP fusion proteins synthesized at given times. The same amount (15 µg) of each whole cell lysate was subjected to the analysis. Experiments were performed independently at least three times and the values are expressed with standard deviations. (C) A ‘quasi-prion’ state induced by coaggregation. [PSI+] cells carrying a maintainer Sup35SC plasmid were transformed with plasmids expressing the indicated chimeras (a), and transformants with (b) or without (c) the maintainer Sup35SC plasmid were plated on SC-His-Ura (b) or SC-His (c) supplemented with low adenine (LA).
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