Variant-specific [PSI⁺] Infection Is Transmitted by Sup35 Polymers within [PSI⁺] Aggregates with Heterogeneous Protein Composition

Abstract

The [PSI(+)] prion is the aggregated self-propagating form of the Sup35 protein from the yeast Saccharomyces cerevisiae. Aggregates of Sup35 in [PSI(+)] cells exist in different heritable conformations, called "variants," and they are composed of detergent-resistant Sup35 polymers, which may be closely associated with themselves, other proteins, or both. Here, we report that disassembly of the aggregates into individual Sup35 polymers and non-Sup35 components increases their infectivity while retaining their variant specificity, showing that variant-specific [PSI(+)] infection can be transmitted by Sup35 polymers alone. Morphological analysis revealed that Sup35 isolated from [PSI(+)] yeast has the appearance of short barrels, and bundles, which seem to be composed of barrels. We show that the major components of two different variants of [PSI(+)] are interacting infectious Sup35 polymers and Ssa1/2. Using a candidate approach, we detected Hsp104, Ssb1/2, Sis1, Sse1, Ydj1, and Sla2 among minor components of the aggregates. We demonstrate that Ssa1/2 efficiently binds to the prion domain of Sup35 in [PSI(+)] cells, but that it interacts poorly with the nonaggregated Sup35 found in [psi(-)] cells. Hsp104, Sis1, and Sse1 interact preferentially with the prion versus nonprion form of Sup35, whereas Sla2 and Ssb1/2 interact with both forms of Sup35 with similar efficiency.

Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells

“…6, C-F), including short barrel-like structures (Fig. 6E) that somewhat resembled structures from earlier attempts to visualize natural prion aggregates (51,52). When recovered protein was allowed to sit for several days at room temperature, some longer filamentous structures were observed including helical structures (Fig.…”

“…Sis1, along with other chaperones, is known to be associated with Rnq1 prion aggregates (51), and mutations in SIS1 can affect Rnq1 aggregation (45,46). Yet, no other chaperones were identified by TAPI, implying that among chaperones Sis1 has the strongest binding or a distinct mode of binding to prion aggregates.…”

Prions in Yeast

“…At a molecular level, excess Ssa increases both the size of Sup35 polymers and the proportion of non-aggregated Sup35 (Allen et al 2005). Ssa physically interacts with Sup35 (Allen et al 2005) and was identified as a major non-Sup35 component associated with [PSI + ] aggregates in vivo (Bagriantsev et al 2008). Excess Ssb also antagonizes weak variants of [PSI + ] upon prolonged propagation (Kushnirov et al 2000b;Chacinska et al 2001) or other [PSI + ] variants in the presence of excess Sup35 (Allen et al 2005).…”

“…Ure2 fibers were identified in [URE3] cells overexpressing Ure2 with EM (Speransky et al 2001 (Bagriantsev et al 2008). The fluorescent rings and dots formed in the process of prion induction by overexpressed Sup35-GFP (see Prion induction by overproduction) were shown to be made of fibrils (Tyedmers et al 2010).…”

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

“…129 Moreover, proteins regulating the assembly and disassembly of actin filaments involved in formation of endocytic vesicles, such as for example Sla1, Sla2 and End3 interact with prion aggregates. [64][65][66]130 It was suggested that particularly during de novo formation of the [PSI + ] prion, the misfolded prion determinants, e.g., Sup35 or NM-GFP, associate with the cytoskeleton, which may facilitate local concentration and subsequently conformational conversion 131 of the prion determinants to the prion state. 65,86 A possible molecular link between the actin cytoskeleton and prion aggregates, especially during stress induced prion formation, is the stress-inducible protein Lsb2.…”