Interactions between non-prion and prion domains of Rnq1 direct formation of amyloid vs liquid-like aggregates and create transmission barriers

Abstract Prions are self-propagating protein conformations usually existing as amyloid aggregates. [ PIN + ], a prion form of the Rnq1 protein occasionally found in wild and laboratory yeast strains, facilitates both the de novo formation and destabilization of other yeast prions, and affects aggregation and toxicity of human misfolding disease proteins expressed in yeast. Rnq1 contains a short N-terminus with no confirmed function (the non-prion domain, NPD) and a C-terminus that carries four QN-rich regions and is sufficient for [ PIN + ] formation and maintenance (prion domain, PD). In the current study, a genetic screen identified the NPD T27P mutation that blocks transmission of the [ PIN + ] prion state from wild type Rnq1 (Rnq1 WT ) to mutant Rnq1 T27P . The mutation doesn’t prevent Rnq1 T27P from switching to a prion state when overexpressed in vivo , or from forming amyloid fibers in vitro . Furthermore, like [ PIN + WT ], the newly formed [ PIN + T27P ]s promote the de novo appearance of the Sup35-based prion [ PSI + ]. We conclude that the NPD mutation creates a barrier for prion transmission from [ PIN + WT ] to Rnq1 T27P . Because fluorescence microscopy shows that Rnq1 T27P efficiently joins [ PIN + WT ] aggregates, the barrier is likely due to the inability of Rnq1 T27P to propagate the specific [ PIN + WT ] conformational variant. Indeed, the analysis of [ PIN + T27P ]s resulting from rare transmission events from [ PIN + WT ] indicates that these [ PIN + T27P ]s must undergo conformational adaptation to yield more stable prion variants. Deletion analysis revealed that T27P constrains prion conformations through the first two QN-rich regions within the PD. The finding that Rnq1 T27P -YFP readily forms non-amyloid liquid-like droplets, which Rnq1 WT -YFP does not form, supports the idea that the NPD affects aggregation properties of the PD. We propose that these aggregation properties are essential for Rnq1’s functions, such as controlling aggregation of other proteins. This provides new insight into the role of heterologous proteins and transmission barriers in the origins of protein misfolding diseases. Author Summary Proteins must fold into the right shapes to work properly. Sometimes they fold incorrectly and stick together, forming long fiber aggregates that damage cells. This kind of “protein misfolding” causes human diseases such as Alzheimer’s. Certain yeast proteins behave similarly, making them useful to study this process. We investigate a yeast protein called Rnq1, which has a region that helps it misfold into fibers. These fibers can also cause other, unrelated proteins to misfold. We found that a mutation in a different part of Rnq1— outside the aggregation region — reduces the ability of non-mutant Rnq1 fibers to convert mutant Rnq1 into growing fiber aggregates. We also identified which section of the aggregation region is affected by this mutation. Interestingly, although the rarely converted mutant aggregates grow poorly at first, they can eventually “adapt” into a shape that grows better. The same mutation also pushes Rnq1 to form liquid-like droplets instead of fibers. Our findings show that the non-aggregating part of Rnq1 controls how Rnq1 aggregates, and, consequently, the appearance and elimination of aggregates formed by other proteins. Our work also helps explain how barriers to misfolded protein growth can be overcome, which is relevant to understanding human protein misfolding diseases.