In our study of pathways involved in amyloid formation and dissolution, we have used yeast as our model system with a focus on naturally occurring prions and mammalian huntingtin fragments with expanded polyglutamine repeat region. Interestingly, prion proteins have an intrinsically disordered domains, which are responsible for these proteins to phase separate under energy depletion conditions. The phase separation has been characterized for Sup35 prion protein, but not for the Rnq1and Ure2. We have extended our studies to compare the phase separation of the Rnq1 and Ure2 prion proteins to that of Sup35 under different conditions. Using GFP-labeled prion proteins, fluorescence imaging was used study found that all three prion proteins promote phase transition of the three canonical prion proteins, Rnq1, Sup35, and Ure2. Specifically, we wanted to examine whether the three different prion proteins all respond similarly to change conditions even though aside from the prion domain, their other domains are not conserved. We find that that GFP-labeled Sup35, Ure2, and Rnq1 are form reversible condensates under starvation conditions, even at a neutral internal pH, suggesting that aggregate formation may be a metabolic response to stress. We also show that addition of 2,4-dinitrophenol, a mitochondrial uncoupler, renders this response pH-sensitive. Using image analysis tools, we quantify these results, showing that less than 20 percent of the GFP-labeled prion forms these aggregates. We also show that stress granule marker Pub1, partially colocalizes with condensates of Sup35, but not with condensates of either Ure2 or Rnq1. In addition, we now find that the curing of URE3 by asymmetric segregation is regulated by three chaperones, Hsp42, Sis1, and Hsp70. Hsp42 increases aggregate formation by sequestering the Ure2 foci, whereas Sis1/ Ssa1 work together to dissociate the clumps. In turn, reducing Sis1/Ssa1 levels in the cell leads to curing by asymmetric segregation, but the rate and extent of clumping of Ure2 foci is reduced in an HSP42 deletion strain. Interestingly, the chaperone role of Sis1/Ssa1 in dissociating clumps is independent of their role in severing the prion seeds. The severing of the seeds is dependent on Hsp104, which is activated by the binding of Sis1 and SSa1. Once activated, Hsp104 can then sever the prion seeds. Interestingly, the function of these chaperones in the curing of URE3 prion is very different from their function found for the sorting of non-amyloidgenic misfolded proteins to degradative compartments. In a related project, we are examining the aggregation of huntingtin (Htt) exon 1 fragments in yeast, which has been used as a system for studying Huntingtons disease. In yeast as in mammalian cells, aggregation of Htt fragments is dependent on the length of the polyglutamine repeat region, but unlike mammalian cells, Htt fragment aggregation has been reported to be dependent on the presence of a yeast prion. This, in turn, makes aggregation dependent on Hsp104, which by severing the prion seeds enables them to propagate. However, it is not clear whether Hsp104 has a role that is independent of prion propagation. To investigate this question, we expressed HttQ103 in yeast while simultaneously inactivating Hsp104 with guanidine. In contrast to the numerous foci of varying sizes that form in the presence of prion and active Hsp104, with inactive Hsp104, there were much fewer smaller foci and with further growth of the yeast, the small aggregates coalesced to form a large aggregate. Washing out the guanidine to reactivate the Hsp104 led to the rapid accumulation of numerous HttQ103 foci, suggesting that Hsp104 severs both the large aggregates and the Htt foci, thereby leading to amplification of the Htt foci. Consistent with this role of Hsp104, in yeast with no Hsp104, cells did not accumulate Htt aggregates even after a week, but in cells with no prion, but with Hsp104, cells slowly accumulated HttQ103 over a period of a week. In contrast, in the presence of both prion and Hsp104, the cells all accumulated numerous aggregates within several hours. These results show that prion greatly accelerates the formation of Htt aggregates, which then act as substrates for Hsp104 activity, leading to the formation of still more aggregates.
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