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

Figure 4.

Figure 4

Hsp70 and Hsp40 target NM oligomers and fibres to inhibit fibrillization. (A) NM (2.5 μM) with ATP (5 mM) was rotated for 5 min (80 r.p.m.) in the presence or absence of Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (2.5 μM) (black bars). Alternatively, NM (2.5 μM) with ATP (5 mM) alone was rotated (80 r.p.m.) for 30 min and then Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (2.5 μM) were added. This reaction was continued for 10 min (blue bars). Oligomeric NM was recovered by centrifugation at 436 000 g for 30 min and the amount in the pellet fraction determined. Values represent means±s.d. (n=3). (B) Sup35 (2.5 μM) with ATP (5 mM) was rotated for 1 h (80 r.p.m.) in the presence or absence of either Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (2.5 μM) (black bars). Alternatively, Sup35 (2.5 μM) with ATP (5 mM) alone was rotated (80 r.p.m.) for 1 h and then Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (2.5 μM) were added. This reaction was continued for 10 min (blue bars). Oligomeric Sup35 was recovered by centrifugation at 436 000 g for 30 min and the amount in the pellet fraction determined. Values represent means±s.d. (n=3). (C) NM (2.5 μM) with ATP (5 mM) was rotated (80 r.p.m.) for 0–50 min. At 0, 15 and 45 min, His–tagged Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 were added. At 50 min, reactions were depleted of chaperones using Ni-NTA agarose. Depleted reactions were spotted onto nitrocellulose and probed with anti-oligomer or anti-NM antibody. (D) Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Ydj1 H34Q, Ssa1:Sis1, Ssb1:Ydj1, Ssb1:Ydj1 H34Q or Ssb1:Sis1 (2.5 μM) were incubated in the presence of ATP (5 mM) with Ni-NTA beads (empty) or beads bound to NM–His monomers, oligomers or fibres. Washed beads were eluted and eluates were fractionated by SDS–PAGE and Coomassie-stained or processed for immunoblot (to detect Sis1). (E) Ssb1 or Ssa1 (1 μM) were incubated for 30 min at 25°C with or without Ydj1 or Ydj1 H34Q (1 μM) in the presence or absence of NM oligomers or fibres (2.5 μM). The amount of ATP hydrolysis was then determined. Values represent means±s.d. (n=3). (F) Ssa1, Ssb1, Ydj1, Sis1, Ssa1:Ydj1, Ssa1:Sis1, Ssb1:Ydj1 or Ssb1:Sis1 (2.5 μM) were incubated in the presence of ATP (5 mM) with Ni-NTA beads (empty) or beads bound to His–Sup35 monomers, oligomers or fibres. Washed beads were eluted, and eluates were fractionated by SDS–PAGE and Coomassie-stained. (G) NM–His monomers, oligomers or fibres were transformed into [psi−] cells. The proportion of [PSI+] colonies was then determined. Values represent means±s.d. (n=3).