Figure 9.

Soluble α-synuclein A53T protein directly binds ER/Golgi SNAREs and inhibits 4-helix bundle assembly in vitro. (A) Glutathione beads preloaded with GST or GST-α-syn A53T were incubated with one of three concentrations of each individual purified, soluble SNARE as indicated along the top edge of the top panel (18 binding reactions in total). Beads were washed extensively with buffer and then analyzed by SDS-PAGE, ponceau staining, and immunoblotting for bead-retained SNAREs using the antibodies listed along the left edge. Ponceau staining for the same set of blots is shown in Supplemental Figure S5A. (B) Quantitation of the binding experiment from A. (C) Glutathione beads preloaded with GST-membrin were incubated with the soluble proteins listed above the blots (see Materials and Methods for detailed conditions). After a 1-h binding incubation, beads were sedimented and washed extensively. The presence of syntaxin 5 was determined by quantitative immunoblotting of the washed beads (top blot) and the supernatant from the first centrifugation (bottom blot). (D) Quantification of bead-bound syntaxin 5 from two experiments like that shown in C. Binding of syntaxin 5 to control GST beads is also included, but for simplicity was not shown in C. Error bars indicate SE where exceeding symbol size. Ponceau stain of the blot in C can be found in Supplemental Figure S5B. (E) Purified soluble sec22b, syntaxin 5, membrin, and rbet1 were coincubated with either purified soluble GST or GST/α-syn A53T, as indicated with “+” and “−” along right edge. After a 4-h ice incubation with all five purified proteins (see Materials and Methods for detailed conditions), the mixture was fractionated by Superdex 200 chromatography. Selected column fractions, listed along the top edge, were analyzed by SDS-PAGE and immunoblotting using the antibodies listed along the left edge. Positions of globular proteins of known molecular size or blue dextran (“Vo”) are indicated with arrows above the fraction numbers. (F) Coomassie-stained SDS-PAGE gel analysis of GST and GST/α-syn A53T preparations in the same proportions used in the preincubations in parts C and E. An asterisk marks the α-syn A53T protein band.