The types of [PIN+] prions and liquid-like aggregates formed by the yeast protein Rnq1 are controlled by interactions between its non-prion and prion domains
Authors: Derkatch Irina L. , Kadnar Michele L. , Liebman Susan W. , Andrade Monica , Fomitchova Anna P. , Maldonado Dania M. 
Published: January 2025

Prions are self-propagating protein conformations usually existing as amyloid aggregates. [PIN+], a prion form of the Rnq1 protein frequently found in wild and laboratory yeast strains, facilitates both thede novoformation and destabilization of other yeast prions, and affects aggregation and toxicity of human misfolding disease proteins expressed in yeast. Rnq1 consists of a short N-terminal domain with no confirmed function (the non-prion domain, NPD) and a C-terminal QN-rich domain sufficient for [PIN+] formation and maintenance (prion domain, PD). The prion domain carries four discrete QN-rich regions. None of these are essential for stable [PIN+] prion maintenance, and any two are sufficient. In the current work, a genetic screen identified the T27P mutation in the beginning of the NPD that blocks transmission of the [PIN+] prion state from wild type Rnq1 (Rnq1WT) to mutant Rnq1T27P. However, the mutation does not prevent overexpressed Rnq1T27Pfrom taking on a prion conformationin vivoor the assembly of Rnq1T27Pamyloid fibersin vitro. Furthermore like [PIN+WT], the newly formed [PIN+T27P] promotes thede novoappearance of the Sup35-based prion [PSI+]. This indicates that the mutation specifically creates a barrier for the transmission of the prion state from [PIN+WT] to Rnq1T27P. Because fluorescence microscopy shows that Rnq1T27Pefficiently joins [PIN+WT] aggregates, the barrier is likely due to the inability of Rnq1T27Pto propagate the [PIN+WT] conformation once Rnq1WTis gone. Indeed, the analysis of [PIN+T27P] cultures resulting from rare transmission events from [PIN+WT] confirmed that initially the [PIN+T27P] prions are very unstable and are lost from most mitotic progeny. However, studies of [PIN+T27P] lineages for >100 generations revealed that such prions gradually stabilize. While some [PIN+T27P] lineages reach stability indistinguishable from that of [PIN+WT], others reach different levels of stability that do not change upon further propagation, indicative of the formation of distinct [PIN+T27P] prion conformations. This supports the idea that the underlying reason for the transmission barrier is that Rnq1T27Pis unable to efficiently propagate the specific [PIN+WT] prion strain conformation but can undergo conformational adaptation even in non-selective conditions. Such conformational adaptation was previously observed when a prion was transmitted from [PIN+WT] to Rnq1 fragments lacking one or more QN-rich regions in the PD. Indeed, we now find that deletion of the first QN-rich region of the PD considerably reduces the transmission barrier created by the T27P mutation, indicating that the NPD can create conformational constraints on a particular region of the PD. Another finding supporting the idea of the effect of the NPD on aggregation properties of the PD is that Rnq1T27Preadily forms non-amyloid liquid-like droplets which Rnq1WTdoes not form. Based on comparison of the Rnq1 homologs from other species, the T27P mutation is located in one of two conserved regions of the NPD, the other region being the interaction site for the Sis1 chaperone involved in [PIN+] maintenance and toxicity upon Rnq1 overexpression. The current findings, together with prior data, suggest that the entire Rnq1 protein contributes to its aggregation and prionization properties, and that these properties are essential for Rnq1 function, possibly in controlling the aggregation of other proteins.