Overexpressed Pin4C sequesters Hsp104 from the cytoplasm.

<p>(A) Overexpressed Pin4C sequestered Hsp104-GFP to colocalize with Pin4C-DsRed aggregates. Representative images of cells with endogenous Hsp104 tagged with GFP without or with 16 hrs of induction of pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> are shown. (B) Overexpressed Pin4C binds to Hsp104. The interaction between Hsp104 and overexpressed Pin4C was assayed in strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) following overnight overexpression of Pin4C-DsRed. 60 µg of total protein was loaded as the “lysate”. The Pin4C-DsRed complex immunocaptured with anti-DsRed from 500 µg of total protein was loaded as “eluate”. The same membrane was immunoblotted (IB) with anti-DsRed, then with anti-Hsp104, and re-probed with anti-Pgk1 as a control. No co-immunocapture of endogenous Pgk1 with Pin4C was detected, implying that Hsp104 was specifically immunocaptured with the Pin4C complex. The slightly slower migration of Hsp104 in the “eluate” relative to its migration in the “lysate” is probably due to the different buffers used during immunocapture. (C) Overexpression of Hsp104 ^T160M suppresses curing of strong [<i>PSI</i>⁺] by Pin4C. Transformants with pHR81<i>GAL</i>-<i>PIN4C</i> and pRS413<i>GAL</i>-<i>HSP104^T160M</i> (↑Pin4C, ↑Hsp104^T160M); or with pHR81<i>GAL</i>-<i>PIN4C</i> and empty vector pRS413<i>GAL</i> (↑Pin4C); or with pRS413<i>GAL</i>-<i>HSP104^T160M</i> and pHR81<i>GAL</i> (↑Hsp104^T160M); or with both empty vectors pHR81<i>GAL</i> and pRS413<i>GAL</i> (↑vectors) were selected on plasmid selective glucose medium, replica-plated to plasmid selective inducing galactose medium, and then 10-fold serially diluted (10⁵→10⁰ cells from left to right) and spotted onto glucose YPD medium where expression of Pin4C and Hsp104^T160M is turned off. There was no growth inhibition in cells overexpressing Pin4C and Hsp104^T160M compared to those overexpressing Pin4C alone when spotted on a galactose plate (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#pgen.1003236.s005" target="_blank">Figure S5</a>). [<i>PSI⁺</i>] loss was scored by the appearance of red [<i>psi</i>⁻] colonies.