FIGURE 1: Effect of genetic modifiers of toxic HDEL-Aβ₄₂ on Aβ₄₂-RF oligomer formation and Aβ₄₂-MRF aggregates into toxic SDS-stable oligomers in yeast.

(A) Overexpressed Yap1802 but not other previously described [24] HDEL-Aβ₄₂ modifiers dramatically enhanced the translational termination factor activity of Aβ₄₂-RF. Aβ₄₂-RF expressing cells (CUP1::Aβ₄₂-RF) lacking chromosomal SUP35 were transformed with a plasmid carrying the indicated gene under control of the GAL1 promoter. Ten-fold serial dilutions of transformants are shown on plasmid selective non-inducing media (SD-Ura); galactose plasmid selective media (SGal-Ura) to turn on each gene (↑ORF); +Ade medium containing copper to overexpress Aβ₄₂-RF (↑ORF ↑Aβ₄₂-RF); the identical -Ade medium SGal-Ura-Ade+Cu²⁺ (-Ade ↑ORF ↑Aβ₄₂-RF) to determine translational termination factor activity as read-through of the ade1-14 nonsense mutant. The Aβ₄₂-RF overexpressed in cells was shown to have reduced translational termination factor activity as cells grew on -Ade due to aggregation of the fusion protein into small oligomers (Aβ₄₂-RF-e. v.). However, the translation termination factor activity was retained in yeast cells overexpressing Aβ₄₂m2-RF (Aβ₄₂ aggregation-deficient mutant) or YAP1802, due to the absence of oligomer formation, resulting in reduced growth on -Ade (Aβ₄₂m2-RF-e. v.).

(B) Yap1802 suppression of Aβ₄₂-RF oligomerization by immunoblot analysis. Total cell lysates were prepared from 2 independent transformants of an Aβ₄₂-RF strain carrying YAP1802 (+) or an empty vector (-) plasmid. Both Aβ₄₂-RF and YAP1802 were overexpressed (SGal-Ura-Ade+Cu²⁺) prior to lysis. Immunoblots were probed with anti-Sup35 RF to evaluate the level of oligomers and monomers. ↑Yap1802 indicates overexpression of YAP1802. The identification of bands as Aβ₄₂-RF dimers and monomers was determined by the estimated sizes of the bands in the immunoblot.