Fig. 2.

Coaggregation of heterologous Sup35 proteins in S. cerevisiae. (A) The S. cerevisiae [PSI⁺] strain simultaneously expressing both endogenous (Sup35_SC) and heterologous (Sup35_SP or Sup35_SB) proteins shows all of the Sup35-reacting material in the pellet (P) after centrifugation at 39,000 × g, whereas the isogenic [psi⁻] strain retains a fraction of Sup35 in the supernatant (S). Shift of Sup35_SB to pellet can be monitored directly because of its lower molecular weight, compared with Sup35_SC. T, total lysate. (B) The chimeric Sup35NM_SP-GFP protein, expressed from the P_CUP1 promoter in the presence of background levels (2 μM) of CuSO₄, shifts to pellet together with the endogenous Sup35_SC protein in the [PSI⁺] extract, in contrast to the [psi⁻] extract. Designations are as in A. (C–F) The GFP-tagged NM fragments of Sup35_SC (C), Sup35_SP (D), and Sup35_SB (E), but not the GFP-tagged Sup35NM fragment of P. methanolica (Sup35NM_PM-GFP, F), colocalize with the aggregated clumps of RFP-tagged Sup35_SC in the S. cerevisiae [PSI⁺] cells. GFP- and RFP-tagged constructs were expressed from the P_CUP1 and P_GAL promoters, respectively, in the Gal+Raf medium supplemented with 150 μM CuSO₄. In each case, >100 cells containing both GFP and RFP aggregates were scored, and the percentage of cells with colocalization is shown. Scale bars are indicated.