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. 2007 Jan 19;365(3):773-82.
doi: 10.1016/j.jmb.2006.10.069. Epub 2006 Oct 25.

"Prion-proof" for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+]

Basant K Patel  1 , Susan W Liebman
Affiliations

"Prion-proof" for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+]

Basant K Patel et al. J Mol Biol. .

Abstract

Prions are self-propagating, infectious protein conformations. The mammalian prion, PrP(Sc), responsible for neurodegenerative diseases like bovine spongiform encephalopathy (BSE; "mad cow" disease) and Creutzfeldt-Jakob's disease, appears to be a beta-sheet-rich amyloid conformation of PrP(c) that converts PrP(c) into PrP(Sc). However, an unequivocal demonstration of "protein-only" infection by PrP(Sc) is still lacking. So far, protein only infection has been proven for three prions, [PSI(+)], [URE3] and [Het-s], all of fungal origin. Considerable evidence supports the hypothesis that another protein, the yeast Rnq1p, can form a prion, [PIN(+)]. While Rnq1p does not lose any known function upon prionization, [PIN(+)] has interesting positive phenotypes: facilitating the appearance and destabilization of other prions as well as the aggregation of polyglutamine extensions of the Huntingtin protein. Here, we polymerize a Gln/Asn-rich recombinant fragment of Rnq1p into beta-sheet-rich amyloid-like aggregates. While the method used for [PSI(+)] and [URE3] infectivity assays did not yield protein-only infection for the Rnq1p aggregates, we did successfully obtain protein-only infection by modifying the protocol. This work proves that [PIN(+)] is a prion mediated by amyloid-like aggregates of Rnq1p, and supports the hypothesis that heterologous prions affect each other's appearance and propagation through interaction of their amyloid-like regions.

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Figures

Figure 1
Figure 1. Scheme of in vitro transformation of [pin−] yeast
Amyloid aggregates of Rnq1p-(132–405) or crude cell extracts from yeast bearing variants of [PIN+] were introduced by co-transformation into [pin−][psi−] cells along with the LEU2 plasmid pGALSUP35. Plasmid bearing transformants were selected on leucine-less medium (see Materials and Methods). Some of the Leu+ transformants also took up the infective [PIN+]-specific Rnq1p aggregates which converted these cells to [PIN+] by sequestering the endogenous Rnq1p into prion aggregates. We used the fact that [PIN+] facilitates the appearance of [PSI+] to score the transformants for [PIN+]. The Leu+ transformants were grown for 48 h on plasmid selective galactose medium to over-express Sup35p, which causes the appearance of [PSI+] in the presence of [PIN+]. Induction of [PSI+] as a measure of [PIN+] infection was assessed by growth on adenine-less medium (SC-Ade) and confirmed by curability by GuHCl (see Materials and Methods).
Figure 2
Figure 2. Variant-specific infectivity of crude cell extracts from [PIN+] and [RPS+] yeast
(A) Crude cell extracts containing 300 µg protein from yeast bearing the four [PIN+] variants (low, medium, high or very high) or from [pin−] yeast were introduced into [pin−][psi−] cells via co-transformation with pGALSUP35. Plasmid transformants (Transformant) and the yeast from which the extracts used as seed were made (Seed) were assayed for [PIN+] by determining if [PSI+] could be induced by Sup35p over-expression. [PIN+] variants were distinguished by the efficiency with which they promote [PSI+] appearance as detected by growth on adenine-less medium (SC-Ade). (B) Crude cell extracts from [RPS+] or [rPS−] yeast containing 300 µg protein were co-transformed with pGALSUP35 into [rPS−] yeast and Leu+ transformants (Transformant) and the yeast from which the extracts used as seed were made (Seed) were assayed for [RPS+] by patching single colonies on complex medium (YPD) and examining color. [RPS+] and [rPS−] yeast are respectively white and red.
Figure 3
Figure 3. Kinetics of in vitro aggregation of recombinant Rnq1p-(132–405)
(A) Effect of agitation on the rate of aggregation of 110 µM Rnq1p-(132–405) at 27 °C measured by thioflavin-T dye binding. Samples were incubated either without or with 30 sec agitation every 3 min. (B) Effect of protein concentration on the rate of Rnq1p-(132–405) aggregation monitored with agitation at 27 °C (C) Effect of pre-formed seed concentration on the aggregation kinetics of 110 µM Rnq1p-(132–405) agitated at 27 °C (D) Effect of increasing temperature on the rate of aggregation of Rnq1p-(132–405) monitored with agitation. Thick lines represent 70 µM and the thin lines indicate 105 µM of the protein.
Figure 4
Figure 4. Characterization of in vitro-made aggregates (27 °C) of Rnq1p-(132–405)
(A) Secondary structure in sonicated Rnq1p-(132–405) aggregates (●) monitored by far UV circular dichroism compared with non-aggregated protein maintained in 9M Urea (□). (B) Stability of Rnq1p-(132–405) aggregated (55 µM) to increasing GuHCl concentration 22 incubated for 90 min at room temperature. (C) Proteinase K digestion of the in vitro-made Rnq1p-(132–405) aggregates compared to the monomeric protein, incubated at 37 °C for 30 min. (D) Western blot showing stability of the aggregates of Rnq1p-(132–405) to 2% SDS monitored by electrophoresis on 1.5% agarose gel and probed by anti-Rnq1 type-II antibody. Lane I, non-aggregated protein; Lane II, in vitro-made aggregates.
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