Volume 8 issue 11 November 2007
In vitro fusion catalyzed by the sporulation specific t-SNARE light chain Spo20p is stimulated by phosphatidic acid
Song Liu, Kirilee A. Wilson, Travis Rice-Stitt, Aaron M. Neiman, and James A. McNew

Supplemental Figure 1: Unit comparison for in vitro fusion data. Conversion of percent maximum fluorescence to rounds of fusion. (Top) The relative fluorescence data normalized to percent maximum fluorescence as shown in Figure 4A. (Bottom) The normalized percent of maximum fluorescence signal is then converted to Rounds of Fusion utilizing the equation Y = (0.49666*e^(0.036031*X)) - (0.50597*e^(0.053946*X)). The conversion of normalized percent of maximum fluorescence signal to "Rounds of fusion", which can be more precisely stated as "fold lipid diluition", utilizes a calibration curve generated with lipid mixtures containing prediluted fluorofores (³³). Given the assumption of equal liposome size and equal propensity of all liposomes in the population to fuse, "fold lipid dilution" is equal to "rounds of fusion".

³³. Parlati F, Weber T, McNew JA, Westermann B, Sollner TH, Rothman JE. Rapid and efficient fusion of phospholipid vesicles by the alpha-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc Natl Acad Sci U S A 1999;96(22):12565-12570.

Figure 1 (.jpg)

Supplemental Figure 2: PA does not increase the rate of Sso1p t-SNARE complex formation. Sso1p alone was reconstituted into PCPS or 15%PA liposomes and then incubated with 3 fold excess Spo20c for 12, 24 and 36 hours. The liposomes were floated in a step gradient to remove unbound proteins and the binding was tested by SDS PAGE. Although the amount of Spo20c-Sso1p binding increased by time, we did not see significant more binding for PA liposomes than PCPS liposomes.

Figure 2 (.jpg)