α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin
Figure 2
α-SNAP-M105I binds syntaxin with higher affinity than wild type α-SNAP.
A, 4 µg recombinant α-SNAP (wild type and mutants) were immobilized by binding to the surface of polypropylene microcentrifuge tubes for 20 min at 20°C. Excess protein was removed and non-specific binding sites were blocked as described under “Materials and Methods” before incubating with 2 µg recombinant NSF for 10 min at 4°C. All tubes were then washed and the immobilized proteins were recovered by boiling for 5 min in Laemmli sample buffer. Proteins were resolved on 10% SDS-polyacrylamide gels and analyzed by anti-NSF (top) and anti-α/β-SNAP (loading control, bottom) Western blot. On the far right lane we ran recombinant NSF as a control. Shown is an experiment representative of three repetitions. B, left, syntaxin1 (0.9 µM) was incubated with 5 µM wild type α-SNAP, 5 µM α-SNAP-(160–295), 5 µM α-SNAP-L294A, or 2.5 µM α-SNAP-M105I in a buffer containing 5 mM MgCl2 for 2 h prior to addition of 0.3 µM NSF together with 2 mM ATP or ATP-γ-S. After an additional 1 h at 30°C, syntaxin was collected by inmunoprecipitation as described under “Materials and Methods.” Precipitated protein complexes were separated on 10% SDS-polyacrylamide gels and immunoblotted with the anti-α/β-SNAP (top) or the monoclonal anti-syntaxin1 (bottom) antibodies. LC, immunoglobulin light chain, HC, immunoglobulin heavy chain,* indicates the electrophoretic mobility of α-SNAP-(160–295). Mr standards (×103) are indicated on the left. Shown is an experiment representative of three repetitions. Right, densitometric analysis of Western blots for α-SNAP (mean ± SEM, n = 3) showing the fraction of α-SNAP coimmunoprecipitated with syntaxin normalized to the amount of syntaxin in each sample. Gray bars, control amount of syntaxin-bound α-SNAP when ATP hydrolysis was prevented (ATP-γ-S lanes, set to 100% for each protein version); black bars, syntaxin-bound α-SNAP after NSF/ATP-driven disassembly expressed as a percentage of the amount precipitated when ATP hydrolysis was prevented. ** p<0.01 for α-SNAP wt ATP vs ATP-γ-S and * p<0.05 for α-SNAP-M105I ATP vs ATP-γ-S (Student's t-test for single group mean); p<0.01 for α-SNAP-M105I ATP vs α-SNAP wt ATP (Student's t-test for unpaired comparison). C, Syntaxin1 was incubated with the indicated concentrations of wild type α-SNAP or M105I as in B, except that NSF and ATP were omitted, and samples were processed for syntaxin immunoprecipitation and Western blot. Shown is a blot (out of four repetitions) probed for α-SNAP (top) and syntaxin (bottom). Right, densitometric analysis of Western blots including that depicted in C (mean ±S.E.M., n = 4) showing the maximal amount of α-SNAP coimmunoprecipitated with syntaxin as a function of the initial concentration added to each reaction mixture. Each value was normalized taking into account syntaxin's densitometric signal in the corresponding lane.