Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism
Figure 4
Rnq1 aggregates colocalize with Sup35 aggregates when [PSI+] is induced de novo.
A. Colocalization of Sup35 newly appearing vs. mature [PSI+] dots with normal levels of Rnq1. Sup35-RFP was overexpressed (p1678) in [PIN+][psi-] cells with the plasmid p1730 expressing Rnq1-GFP from its own promoter by growth in 2% Gal (top). Gray arrows show additional Sup35 dots that did not overlap Rnq1 and that are away from the vacuole. The middle panel (magnified ā¼2X) shows an Rnq1-GFP dot colocalized with Sup35-RFP dot (white arrow) located near the vacuole stained with FM4-64 (red circle). [PIN+] cells with established [PSI+] were grown in 0.05% Gal for 3ā4 h to stain mature [PSI+] dots marked by Sup35NM-YFP (bottom). These dots showed partial colocalization (enlarged box) with [PIN+] dots marked by Rnq1-CFP. Blue arrows indicate partially colocalized Rnq1 dots; yellow arrows indicate non-colocalized additional Rnq1 dots. B. Colocalization of Sup35 rings with normal levels of Rnq1 expressed from a plasmid. Sup35-RFP was overexpressed from p1678 in [PIN+][psi-] cells with the CEN plasmid p1730 expressing Rnq1-GFP from its own promoter by growth in 2% Gal. Sup35-RFP rings colocalized with Rnq1-GFP rings after 24 h. C. Colocalization of Sup35 rings with normal levels of Rnq1 expressed from an integrated construct. Rnq1 tagged with CFP, integrated into genomic TRP1 was expressed from its own promoter in [PIN+] cells overexpressing Sup35NM-YFP from p1753, by growth in 2% Gal for the indicated times. D. Visualization of [PIN+] aggregates during [PSI+] induction with overexpressed Rnq1 and Sup35. Rnq1-YFP was co-overexpressed with Sup35NM respectively from p1728 and p1893, by growth in 2% Gal for 24 h. Rnq1-YFP formed lines and mesh-like aggregates in the presence of [PIN+] and Sup35NM overexpression, but remained diffuse in [pin-]. In the absence of Sup35NM overexpression, [PIN+] contained only multiple dots of Rnq1-YFP. E. Visualization of [PIN+] aggregates during Sup35 overexpression with normal levels of Rnq1. Untagged Sup35NM (p2036) was overexpressed in [PIN+] RNQ1-CFP integrants in 2% Gal for 48 h. Rnq1-CFP displayed ring/line-like structures in the presence of Sup35NM overexpression in 10% of [PIN+] cells (4 trials, each with nā350).