Effect of Charged Residues in the N-domain of Sup35 Protein on Prion [PSI+] Stability and Propagation

Background: The prion domain (PrD) of Sup35p can aggregate to form the [ PSI + ] prion. Results: Introduction of charged lysine residues ( sup35 KK ) in the Sup35p PrD alters prion properties. Conclusion: Some s up35 KK alleles lead to the formation of new prion variants. Significance: Establishment of molecular interactions influencing [ PSI + ] prion stability and maintenance is a step toward an understanding of prion folding. Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register β-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [ PSI + ] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles ( sup35 KK ) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [ PSI + ] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [ PSI + ] prion. This suggests that the critical part of the parallel in-register β-structure for the studied [ PSI + ] prion variant lies in the first 63–69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35 KK alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants.