It may be proposed that some Sup35 amyloid structures possible in vitro would not propagate in vivo, being harmful to cells or nonheritable. Variants of [PSI+] that are too strong would interfere with the cell viability due to the lack of soluble Sup35. At the other extreme, polymers may be fragmented too rarely, which would interfere with their heritability. Such unconventional Sup35 polymer variants may be uncovered in cells with altered Sup35 levels and/or structure.
Overproduction of Sup35 in cells with conventional [PSI+
] causes severe growth inhibition.49
This may be related to the impairment of translation termination due to both the reduced levels of soluble Sup35 and titration of the Sup45 (eRF1) partner termination factor by functionally inactive Sup35 polymers. Any [PSI+
] compatible with Sup35 overproduction would represent a new prion variant not existing under standard conditions.
Two such novel types of [PSI+
] were obtained in a [PIN+
] strain overproducing Sup35: (1) [PSI+
] compatible with Sup35 overproduction, but stable at standard Sup35 levels; (2) [PSI+
] existing only at increased Sup35 levels. In addition, a category of Sup35 amyloid-like polymers was discovered, which does not manifest itself as [PSI+
Surprisingly, these polymers were present in cells before the selection for suppressor phenotype, as revealed by SDD-AGE. Cells containing these polymers did not show suppressor effect due to significant levels of soluble Sup35. About 15% of Sup35 was soluble, which, accounting for 20-fold overproduction, exceeded three-fold the Sup35 levels in [psi-
] cells. Increased levels of soluble Sup35 indicate its inefficient polymerization, which may be related to the size of Sup35 polymers increased several-fold compared to conventional [PSI+
]. In cells with any given Sup35 levels an increase in polymer size would mean a proportional decrease in the number of Sup35 polymers and polymerization speed.
Another property of these polymers was poor heritability. Their appearance required the presence of [PIN+] determinant. Furthermore, [PIN+] elimination via disruption of RNQ1 eliminated the amyloid-like Sup35 polymers in the cells which already have possessed them. Thus, in contrast to [PSI+] polymers, which propagate very stably in the absence of [PIN+], these polymers are not heritable. This may be related to small number of these polymers due to their inefficient fragmentation, which is evident from their large size. Then, the only reason for existence of these polymers is their efficient appearance de novo. This is likely to occur via seeding by Rnq1 polymers, since about 1/5 of total Rnq1 was bound to Sup35 polymers. This bond was resistant to cold SDS, and thus these Rnq1 and Sup35 belonged to the same polymers. It appears unlikely that Rnq1 was dispersed along the Sup35NM polymers, since homotypic polymerization should be much more efficient than heterotypic. More probably, this Rnq1 represented Rnq1 prion seeds attached to the Sup35NM polymers, which they initiated.
Finally, the nonheritable Sup35 amyloid-like polymers appear with very high frequency compared to its prion variants. A clear inverse correlation between the frequency of appearance and the “strength” was observed: stronger variants appeared less frequently.50
The reason for this correlation is not fully clear. It may be presumed that this is due to the preferential survival of prion seeds corresponding to weaker [PSI+
], because they are less recognizable by the cellular chaperones. In this connection, it is noteworthy that among the Sup35 fibers spontaneously formed in vitro, a significant proportion apparently belongs to the prion type, since these fibers could transform yeast cells to the [PSI+
Thus, Sup35 forms amyloid variants covering the full spectrum of related phenotypes, from highly stable strong suppressor [PSI+
] to nonheritable phenotypically undetectable polymers. The key parameter, distinguishing these variants is the frequency of polymer fragmentation, which is highest in strong [PSI+
] polymers and lowest in nonheritable amyloid-like polymers. The ability of Sup35 to form both prions and nonheritable amyloids convincingly supports the idea that prion and amyloid phenomena are related. Notably, another amyloid-forming protein does not make full spectrum of variants: the Sup35 fusion protein with its prion-forming domain replaced with a stretch of 66 glutamine residues can form SDS-insoluble amyloid-like polymers, but is unable to form prions.50