Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion
Figure 4
The SNARE Complexes Remain in syx3–69 Mutant Flies at the Restrictive Temperature
(A and B) The SDS-resistant complex is not obviously affected in homozygous syx3–69 mutant flies at restrictive temperatures. A representative Western blot shows the syntaxin 1A monomer, the 7S SNARE complex, and the multimeric complex obtained from heads of the wild type (+/+) and the syx3–69 mutant (A). A control for total protein loaded is illustrated by the intensity of tubulin (bottom). Histograms of ratios of the 7S and multimeric complexes to monomer in wild-type (+/+) and syx3–69 mutant flies are shown in (B). Unless specifically noted, the complex-to-monomer ratio is normalized to that of the wild type at room temperature in this and other SNARE complex histograms. The SNARE complex was extracted from flies either at room temperature (∼22 °C) or after exposure to 38 °C for 20 min, as described above [21].
(C) An example of Western blots showing the SNARE complex in the wild-type (+/+), comatose (comttp7), and syx3–69 mutant flies. The SNARE complex accumulates in the comttp7 mutant, likely as a result of a block of the NSF ATPase activity at the restrictive temperature. Note that even though relatively less protein was loaded in the syx3–69 lanes (as judged by the intensity of the syntaxin band), the 7S complex remains in paralyzed syx3–69 flies.