Fig. 4.

The Syt7 C₂A domain dominates binding of Syt7 to membranes and clusters liposomes. (A) Titrations of 20 nM Syt7 C₂A domain, C₂B domain and C₂A*B fragment (the * in C₂A*B denotes that the fluorescent probe is attached to C260 of the C₂A domain) with liposomes lacking PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (B) The same titrations shown in A but changing the x axis to better show the points of the titrations obtained at low liposome concentrations. (C) Liposome titrations of 20 nM WT Syt7 C₂AB fragment and mutant fragments where the Ca²⁺-binding sites of the C₂A domain (C₂aB*) or C₂B domain (C₂A*b) were disrupted with D225A,D227A,D233A or D357A,D359A mutations. All of the data in A–C were fit to a Hill function. (D–F) Distribution of particle size measured by DLS on samples containing liposomes alone (D) or liposomes in the presence of the Syt7 C₂A domain (E) or Syt7 C₂B domain (F). In E and F, the diagrams show superpositions of data obtained in the absence (green bars) or presence (red bars) of Ca²⁺.