Hsp40/JDP Requirements for the Propagation of Synthetic Yeast Prions

Abstract

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core “prion fragmentation machinery” includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These “polyQX” PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1′s known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein–protein interactions with individual yeast prion-forming proteins.

Human J-Domain Protein DnaJB6 Protects Yeast from [PSI+] Prion Toxicity

“…To confirm effectiveness of doxycycline repression of Sis1 expression in our hands, we monitored abundance of Sis1 by Western blot ( Figure 3 E). Exposure to doxycycline for 28 h reduced the amount of Sis1 to less than 3% of that of untreated cells, which is comparable to earlier data [ 10 , 44 ].…”