[PSI+] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast

Abstract

[PIN+] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)-rich C-terminal domain is necessary for the propagation of [PIN+], whereas the N-terminal region is non-Q/N-rich and considered the nonprion domain. Here, we isolated numerous single-amino-acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI+] propagation in the [PIN+] state, but not in the [pin-] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi-denaturing detergent-agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI+] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI+]-inhibitory rnq1 mutations did not affect [PIN+] propagation itself when over-expressed from a strong promoter, but instead destabilized [PIN+] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α-helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.