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. 2008 Oct 2;27(20):2712–2724. doi: 10.1038/emboj.2008.194

Figure 6.

Figure 6

Hsp70 and Hsp40 modulate the prion-remodelling activities of Hsp104. (A) Unseeded, rotated (80 r.p.m.) Sup35 (2.5 μM) fibrillization with ATP (5 mM) after 2 h in the presence or absence of Hsp104 (0.03 μM) plus or minus Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (0.25 μM). Fibrillization was assessed by the amount of SDS-resistant Sup35. Values represent means±s.d. (n=3). (B) Sup35 fibres (2.5 μM monomer) were incubated with or without the indicated combination of Hsp104, Ssa1, Ssb1, Ydj1 and Sis1 (2 μM) plus ATP (5 mM). Disassembly was assessed by the amount of SDS-resistant Sup35. Values represent means±s.d. (n=3). (C) Disaggregation of heat-aggregated GFP (0.45 μM) in the presence of the indicated combination of Hsp104, Ssa1, Ssb1, Ydj1 and Sis1 (2 μM) plus ATP (5 mM). Disaggregation was assessed by the amount of GFP fluorescence. Values represent means±s.d. (n=3). (D) Sup35 (2.5 μM) fibres were assembled with rotation (80 r.p.m.) for 8 h with or without various combinations of Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1, Ssb1:Sis1 or Ssa1:Ssb1:Ydj1:Sis1 (0.15 μM) plus ATP (5 mM). Fibres were then collected by sedimentation at 436 000 g for 10 min and treated with SDS–PAGE sample buffer at either 25 or 99°C and processed for immunoblot. (E–G) Sup35 (2.5 μM) fibres were assembled with rotation (80 r.p.m.) for 8 h with or without various combinations of Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1, Ssb1:Sis1 or Ssa1:Ssb1:Ydj1:Sis1 (0.15 μM) plus ATP (5 mM). Hsp104 (0.5 μM or 2 μM) was then added and reactions incubated for a further 30 min. Remodelling was assessed by the amount of SDS-resistant Sup35 (E). Values represent means±s.d. (n=3). (F) Alternatively, products from 2 μM Hsp104 reactions were viewed by EM. Bar, 150 nm. (G) Other reactions were concentrated and transformed into [psi−] cells. The proportion of [PSI+] colonies was then determined. Values represent means±s.d. (n=3).